U.S. patent application number 16/513392 was filed with the patent office on 2019-11-28 for transcription factor genes and proteins from helianthus annuus, and transgenic plants including the same.
The applicant listed for this patent is CONSEJO NACIONAL DE INVESTIGACIONES CIENTIFICAS Y TECNICAS, UNIVERSIDAD NACIONAL DEL LITORAL. Invention is credited to Raquel Lia Chan, Jorge Giacomelli, Jesica Raineri.
Application Number | 20190359996 16/513392 |
Document ID | / |
Family ID | 53052881 |
Filed Date | 2019-11-28 |
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United States Patent
Application |
20190359996 |
Kind Code |
A1 |
Raineri; Jesica ; et
al. |
November 28, 2019 |
TRANSCRIPTION FACTOR GENES AND PROTEINS FROM HELIANTHUS ANNUUS, AND
TRANSGENIC PLANTS INCLUDING THE SAME
Abstract
A polynucleotide having at least 80% sequence identity with the
full-length nucleotide sequence of SEQ ID NO: 1 and substantially
identical polynucleotides; an isolated polypeptide having at least
80% sequence identity with the full-length amino acid sequence of
SEQ ID NO: 2 and substantially identical polypeptides; and
polynucleotides encoding the HaWRKY76 polypeptide and substantially
identical polypeptides are described. Also described are vectors
and recombinant expression cassettes containing the cDNA
polynucleotide, a polynucleotide encoding the HaWRKY76 polypeptide,
or substantially identical polynucleotides. Transgenic plants
containing such expression cassettes, related methods and uses are
also provided.
Inventors: |
Raineri; Jesica; (Santa Fe,
AR) ; Chan; Raquel Lia; (Santa Fe, AR) ;
Giacomelli; Jorge; (Santa Fe, AR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CONSEJO NACIONAL DE INVESTIGACIONES CIENTIFICAS Y TECNICAS
UNIVERSIDAD NACIONAL DEL LITORAL |
Buenos Aires
Santa Fe |
|
AR
AR |
|
|
Family ID: |
53052881 |
Appl. No.: |
16/513392 |
Filed: |
July 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15307333 |
Oct 27, 2016 |
10414807 |
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PCT/GB2015/051269 |
Apr 30, 2015 |
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16513392 |
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61986730 |
Apr 30, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8261 20130101; Y02A 40/146 20180101; C12N 15/8279 20130101;
C12N 15/8273 20130101; C12N 15/8271 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415 |
Claims
1. An isolated polypeptide comprising a sequence having at least
90% sequence identity with the full-length amino acid sequence of
SEQ ID NO: 2, the polypeptide sequence containing at least one of:
(a) proline as the amino acid corresponding to position 270 of the
full-length amino acid sequence of SEQ ID NO: 2; (b) proline as the
amino acid corresponding to position 87 of the full-length amino
acid sequence of SEQ ID NO: 2; (c) proline as the amino acid
corresponding to position 260 of the full-length amino acid
sequence of SEQ ID NO: 2; (d) serine as the amino acid
corresponding to position 123 of the full-length amino acid
sequence of SEQ ID NO: 2; (e) leucine as the amino acid
corresponding to position 14 of the full-length amino acid sequence
of SEQ ID NO: 2; (f) leucine as the amino acid corresponding to
position 22 of the full-length amino acid sequence of SEQ ID NO: 2;
and (g) serine as the amino acid corresponding to position 23 of
the full-length amino acid sequence of SEQ ID NO: 2.
2. The isolated polypeptide of claim 1, wherein the polypeptide
sequence contains proline as the amino acid corresponding to
positions 270 and 87 of the full-length amino acid sequence of SEQ
ID NO: 2.
3. The isolated polypeptide of claim 1, wherein the polypeptide
sequence contains leucine and serine as the amino acids
corresponding to positions 22 and 23, respectively, of the
full-length amino acid sequence of SEQ ID NO: 2.
4. The isolated polypeptide of claim 1, wherein the polypeptide
comprises a sequence having at least 95% sequence identity with the
full-length amino acid sequence of SEQ ID NO: 2.
5. The isolated polypeptide of claim 1, wherein the polypeptide has
the full-length amino acid sequence of SEQ ID NO: 2 or 12.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 15/307,333, filed Oct. 27, 2016, which is a National Phase
application claiming priority to PCT/GB2015/051269 filed Apr. 30,
2015, which claims priority to 61/986,730, filed Apr. 30, 2014, the
entire contents of which are hereby expressly incorporated by
reference in its entirety including, without limitation, the
specification, claims, and abstract, as well as any figures,
tables, or drawings thereof.
BACKGROUND
[0002] Abiotic environmental stresses, such as drought, salinity,
wind, heat, and cold, are major limiting factors of plant growth
and crop yield. Prolonged or continuous exposure to drought
conditions causes major alterations in the plant metabolism that
ultimately lead to cell death and, consequently, losses in crop
yield. High salt content in some soils results in less water being
available for cell intake; thus, high salt concentration has an
effect on plants similar to the effect of drought on plants. Under
freezing temperatures, plant cells lose water as a result of ice
formation within the plant. Because crop damage from abiotic
stresses is predominantly due to dehydration, water availability is
an important aspect of the abiotic stresses and their effects on
plant growth. Losses in crop yield of major crops caused by these
stresses represent a major economic factor and contribute to food
shortages in many underdeveloped countries.
[0003] Most plants have evolved protective mechanisms against
dehydration caused by abiotic stress. However, if the severity and
duration of the abiotic stress conditions are too great, the
effects on development, growth, and yield of most crop plants are
profound. Developing plants efficient in water use is therefore a
strategy that has the potential to benefit human life. Many
agricultural companies have attempted to identify genes that could
confer tolerance to abiotic stress responses, in an effort to
develop transgenic abiotic stress-tolerant crop plants. For
example, the genome of the plant model Arabidopsis was the first to
be sequenced and released by 2000. Although some genes that play a
role in stress responses or efficient water utilization in plants
have been characterized, the characterization and cloning of plant
genes that confer the desired stress tolerance and/or efficient
water utilization characteristics remain largely fragmented and
incomplete.
[0004] The sunflower belongs to the Asteraceae family, whose
members represent 10% of the flowering plants. However, the genome
sequence of this species is largely unknown and the huge quantity
of expressed sequence tags (ESTs) from Helianthus (sunflower)
species available in public databases have not yet been well
explored.
SUMMARY
[0005] Embodiments relate to a polynucleotide having at least 85%
sequence identity with the full-length nucleotide sequence of SEQ
ID NO: 1 or SEQ ID NO: 9, or substantially identical variant
polynucleotides, for example as shown in SEQ ID NO:11. A
polynucleotide according to specific embodiments contains at least
one of: (a) adenine as the nucleotide corresponding to position 817
of the full-length nucleotide sequence of SEQ ID NO: 1 or 11; and
(b) cytosine as the nucleotide corresponding to position 808 of the
full-length nucleotide sequence of SEQ ID NO: 1 or 11. In other
embodiments, the polynucleotide may also contain at least one of
the following: (c) cytosine as the nucleotide corresponding to
position 33 of the full-length nucleotide sequence of SEQ ID NO: 1
or 11; (d) cytosine as the nucleotide corresponding to position 259
of the full-length nucleotide sequence of SEQ ID NO: 1 or 11; and
(e) guanine as the nucleotide corresponding to position 315 of the
full-length nucleotide sequence of SEQ ID NO: 1 or 11.
[0006] Embodiments also relate to vectors comprising a
polynucleotide as described herein, recombinant expression
cassettes comprising a polynucleotide described herein operably
linked to a promoter, transgenic plants comprising such a
recombinant expression cassette, and methods for producing such
vectors, cassettes, and transgenic plants.
[0007] Embodiments also relate to isolated polypeptides having at
least 85% sequence identity with the full-length amino acid
sequence of SEQ ID NO: 2 or 12, or substantially identical
polypeptides, for example as shown in SEQ ID NO:12. Polypeptides
according to specific embodiments contain at least one of: (a)
proline as the amino acid corresponding to position 270 of the
full-length amino acid sequence of SEQ ID NO: 2 or 12; and (b)
proline as the amino acid corresponding to position 87 of the
full-length amino acid sequence of SEQ ID NO: 2 or 12. In
embodiments, a polypeptide disclosed herein may contain at least
one of: (c) proline as the amino acid corresponding to position 260
of the full-length amino acid sequence of SEQ ID NO: 2 or 12; (d)
serine as the amino acid corresponding to position 123 of the
full-length amino acid sequence of SEQ ID NO: 2 or 12; (e) leucine
as the amino acid corresponding to position 14 of the full-length
amino acid sequence of SEQ ID NO: 2 or 12; (f) leucine as the amino
acid corresponding to position 22 of the full-length amino acid
sequence of SEQ ID NO: 2 or 12; and (g) serine as the amino acid
corresponding to position 23 of the full-length amino acid sequence
of SEQ ID NO: 2 or 12.
[0008] Some embodiments relate to polynucleotides that encode a
polypeptide described herein. A vector comprising a polynucleotide
that encodes a polypeptide described herein, a recombinant
expression cassette comprising such a polynucleotide operably
linked to a promoter, transgenic plants comprising such a
recombinant expression cassette, and methods for producing such
products are also provided.
[0009] Some embodiments relate to recombinant expression cassettes
comprising an isolated polynucleotide operably linked to a
promoter, wherein the polynucleotide is a member selected from the
group consisting of: (a) a polynucleotide that encodes the
polypeptide of SEQ ID NO: 2 or a variant thereof, for example SEQ
ID NO: 2; and (b) the polynucleotide of SEQ ID NO: 1 or 9 or a
variant thereof.
[0010] The invention also relates to transgenic plants comprise
such a recombinant expression cassette. The invention further
provides a method of producing a transgenic plant comprising: (a)
introducing into a plant cell such a recombinant expression
cassette; and (b) culturing the plant cell under plant growing
conditions to produce the transgenic plant. The invention also
provides methods for modulating a plant phenotype, for example
increasing yield of a plant under non-stress conditions comprising
introducing and expressing a polynucleotide described herein, for
example the polynucleotide of SEQ ID NO: 1 or a variant thereof,
for example SEQ ID NO: 11, or SEQ ID NO: 9 or a variant thereof.
Also, the invention provides a method for increasing stress
tolerance of a plant to severe and/or moderate stress comprising
introducing and expressing a polynucleotide described herein, for
example the polynucleotide of SEQ ID NO: 1 or a variant thereof,
for example SEQ ID NO: 11, or SEQ ID NO: 9 or a variant
thereof.
[0011] In another aspect, the invention provides the use of a
polynucleotide described herein, for example the polynucleotide of
SEQ ID NO: 1 or a variant thereof, for example SEQ ID NO: 11, or
SEQ ID NO: 9 or a variant thereof or a polypeptide encoded by any
of these polynucleotides in altering a plant phenotype,
specifically in increasing yield of a plant under non-stress
conditions and/or for increasing stress tolerance of a plant to
severe and moderate stress.
BRIEF DESCRIPTION OF THE SEQUENCES
[0012] SEQ ID NO: 1 is an isolated nucleotide sequence (cDNA) of
the HaWRKY76 polynucleotide.
[0013] SEQ ID NO: 2 is an isolated amino acid sequence of the
HaWRKY76 polypeptide.
[0014] SEQ ID NO: 3 is an isolated nucleotide sequence of the
HaT131007971 polynucleotide (as identified in the Helia
database).
[0015] SEQ ID NO: 4 is an isolated amino acid sequence of the
HaT131007971 polypeptide (as identified in the Helianthus annuus
cv. XRQ transcriptome portal).
[0016] SEQ ID NO: 5 is an isolated nucleotide sequence of the
HuCL13748C001 polynucleotide (as identified in the Helia
database).
[0017] SEQ ID NO: 6 is an isolated amino acid sequence of the
HuCL13748C001 polypeptide (as identified in the Helia
database).
[0018] SEQ ID NO: 7 is a conserved motif
[0019] SEQ ID NO: 8 is a conserved motif
[0020] SEQ ID NO: 9 is the HaWRKY76 genomic DNA.
[0021] SEQ ID NO: 10 is the promoter DNA.
[0022] SEQ ID NO: 11 is an isolated variant nucleotide sequence
(cDNA) of the HaWRKY76 polynucleotide
[0023] SEQ ID NO: 12 is an isolated amino acid sequence of the
HaWRKY76 polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a comparison of the amino acid sequence of the
HaT131007971 polypeptide of SEQ ID NO: 4 with the amino acid
sequence of the HaWRKY76 polypeptide of SEQ ID NO: 2.
[0025] FIG. 2 is a comparison of the nucleotide sequence of the
HaT131007971 polynucleotide of SEQ ID NO: 3 with the nucleotide
sequence of the HaWRKY76 polynucleotide of SEQ ID NO: 1.
[0026] FIG. 3 is a comparison of the amino acid sequence of the
HuCL13748C001 polypeptide of SEQ ID NO: 6 with the amino acid
sequence of the HaWRKY76 polypeptide of SEQ ID NO: 2.
[0027] FIG. 4 shows the nucleotide sequence of the HaWRKY76
polynucleotide of SEQ ID NO: 1 with a related sequence.
[0028] FIG. 5 shows the HaWRKY76 expression levels in the roots,
hypocotyls, and cotyledons in 5-day-old sunflower seedlings.
Different letters indicate samples that are significantly different
(p value<0.05). An illustrative photograph of a sunflower plant
in the developmental stage used to take the RNA samples is shown in
the right side.
[0029] FIG. 6A shows the Ha WRKY76 expression levels in 5-day-old
sunflower seedlings, in standard conditions (left) and under severe
drought stress (right) and FIG. 6B shows the expression levels of
sunflower R2 plants subjected to a continuous and severe drought
stress during fifteen days. Different letters indicate samples that
are significantly different (p value<0.05). FIG. 6C is an
illustrative photograph of sunflower plants subjected to severe
water stress and used to take the RNA samples.
[0030] FIG. 7 shows the Ha WRKY76 expression levels of three
homozygous transgenic lines of Arabidopsis transgenic plants
bearing the construct 35S:HaWRKY76.
[0031] FIG. 8A shows root lengths of three homozygous transgenic
lines of Arabidopsis transgenic plants bearing the construct
35S:HaWRKY76 and of WT control plants, and the photograph of FIG.
8B shows roots of the 7-day-old plants grown on Petri dishes.
[0032] FIG. 9 shows the average aerial biomass (weight of detached
rosettes and stems) of 35-day-old plants belonging to three
homozygous transgenic lines of Arabidopsis transgenic plants
bearing the construct 35S:HaWRKY76 and of 35-day-old WT control
plants that were grown in standard conditions. Different letters
indicate samples that are significantly different (p
value<0.05).
[0033] FIG. 10A shows the weight of rosettes detached from plants
belonging to three homozygous transgenic lines of Arabidopsis
transgenic plants bearing the construct 35S:HaWRKY76 and of the WT
control plants grown on poor soil in standard conditions. FIG. 10B
shows the chlorophyll content per mg of rosette leaves of each of
the respective plants. FIG. 10C shows the total protein
concentration of each of the respective plants. Different letters
indicate samples that are significantly different (p
value<0.05).
[0034] FIG. 11 shows the root biomass of 35-day-old plants
belonging to three homozygous transgenic lines of Arabidopsis
transgenic plants bearing the construct 35S:HaWRKY76 and the WT
control plants, all grown on sand irrigated with Hoagland
0.5.times.. Different letters indicate samples that are
significantly different (p value<0.05).
[0035] FIG. 12A shows the average seeds yield of 4 plants of each
genotype grown on poor soil conditions (nutritional deficiency),
FIG. 12B shows the yield per individual plant, and FIG. 12C is a
photograph of the harvested seeds (35S:HaWRKY76 transgenic seeds
and WT control seeds). Different letters indicate samples that are
significantly different (p value<0.05).
[0036] FIG. 13 shows the yields of plants belonging to three
homozygous transgenic lines of Arabidopsis transgenic plants
bearing the construct 35S:HaWRKY76 and of the WT control plants
grown in standard conditions. Different letters indicate samples
that are significantly different (p value<0.05).
[0037] FIG. 14 is a photograph of plants belonging to three
homozygous lines of Arabidopsis transgenic plants bearing the
construct 35S:HaWRKY76 and of WT control plants that have been
subjected to severe drought stress. The photograph is taken 4 days
after the plants were re-watered following the severe drought
stress treatment.
[0038] FIG. 15A is a photograph of the plants belonging to three
homozygous transgenic lines of Arabidopsis transgenic plants
bearing the construct 35S:HaWRKY76 and of WT control plants that
were subjected to drought during the vegetative stage, and FIG. 15C
is a photograph of the respective plants subjected to drought
during the reproductive stage. FIG. 15B shows yields of the
respective plants subjected to drought during the vegetative stage,
and FIG. 15D shows yields of the respective plants subjected to
drought during the reproductive stage. In both treatments, a
nutritional stress (generated by growing the plants on poor soil)
was applied. Different letters indicate samples that are
significantly different (p value<0.05).
[0039] FIG. 16A shows the enhanced yield of Arabidopsis transgenic
plants bearing the construct 35S:HaWRKY76 when drought stress was
applied during the vegetative stage, and FIG. 16B shows that no
significant differences in yield between the genotypes were
observed when the drought stress was suffered during the
reproductive stage. In these experiments, the only stress applied
was drought and other conditions (soil) were standard. Different
letters indicate samples that are significantly different (p
value<0.05).
[0040] FIG. 17A shows the average water added, and FIG. 17B shows
the average yield per plant for each of the three homozygous
transgenic lines of Arabidopsis transgenic plants bearing the
construct 35S:HaWRKY76 and the WT control plants. All of the plants
were grown on soil and normal watering was stopped when the plants
were 25 days old. Thereafter, the minimum quantity of water was
added every two days to maintain the same weight in all pots.
Different letters indicate samples that are significantly different
(p value<0.05).
[0041] FIGS. 18A and 18B show the weight loss of leaves detached
from plants belonging to three homozygous transgenic lines of
Arabidopsis transgenic plants bearing the construct 35S:HaWRKY76
and WT control plants that were well-irrigated (FIG. 18A) and
subjected to moderate drought stress (FIG. 18B). FIG. 18C is an
illustrative photograph of leaves of the respective plants taken
after 8 hours of treatment. Different letters indicate samples that
are significantly different (p value<0.05).
[0042] FIG. 19A shows the percentage of survivors after 25 day-old
plants of three homozygous transgenic lines of Arabidopsis
transgenic plants bearing the construct 35S:Ha WRKY76 and WT
control plants were completely submerged during 6 days and then
placed in standard growth conditions for recovery. FIG. 19B is an
illustrative photograph of the plants taken one week after
recovery. FIG. 19C shows the average seed yields of the recovered
plants.
[0043] FIG. 19D shows the harvested seeds of each genotype. The
number of survivors was lower for the WT genotype plants.
Therefore, the total yield is significantly different (measured as
the average) because only the survivors contribute. Different
letters indicate samples that are significantly different (p
value<0.05).
[0044] FIG. 20A shows the rosette weight of the respective plants
measured 2 and 5 days after starting the treatment conditions
referenced in FIG. 19. FIG. 20B is an illustrative photograph of
the rosettes of the plants taken 5 days after starting the
treatment. FIG. 20C shows the chlorophyll content/mg of rosette
leaves of the respective plants measured 2 and 5 days after
starting the treatment. FIG. 20D shows the content of total soluble
sugars/mg of rosette leaves that was enzymatically determined for
each of the plants. This content was evaluated 2 and 5 days after
starting the treatment. Different letters indicate samples that are
significantly different (p value<0.05).
[0045] FIGS. 21A-C show the content of soluble carbohydrates and
starch in rosettes of 25 day-old plants of three homozygous
transgenic lines of Arabidopsis transgenic plants bearing the
construct 35S:HaWRKY76 and of WT control plants. The 25-day old
plants were submerged during 0, 2, 5 days. FIG. 21A shows the
content of soluble glucose per mg fresh rosette weight of the
respective plants, FIG. 21B shown the sucrose content per mg fresh
rosette weight of the respective plants, and FIG. 21C shows the
starch content per mg fresh rosette weight of the respective
plants. Different letters indicate samples that are significantly
different (p value<0.05).
[0046] FIG. 22A shows the chlorophyll content per mg fresh rosette
weight of 25 day-old plants of three homozygous transgenic lines of
Arabidopsis transgenic plants bearing the construct 35S:HaWRKY76
and of WT control plants. FIG. 22B shows the protein content per mg
fresh rosette weight of the respective plants. The respective
plants were completely submerged during 5 days. Different letters
indicate samples that are significantly different (p
value<0.05).
[0047] FIG. 23A shows the root/aerial biomass ratio of 35 day-old
plants of three homozygous transgenic lines of Arabidopsis
transgenic plants bearing the construct 35S:HaWRKY76 and of WT
control plants. FIG. 23B shows the root protein content of the
respective plants. The respective plants were grown on sand, and on
day 35 the complete root system was collected and weighed.
Different letters indicate samples that are significantly different
(p value<0.05).
[0048] FIG. 24 includes photographic images of 25 day-old plants of
three homozygous transgenic lines of Arabidopsis transgenic plants
bearing the construct 35S:Ha WRKY76 and of WT control plants. The
respective plants were subjected to waterlogging during 5 days, and
transverse sections of stems were performed and stained.
[0049] FIG. 25 shows the average glucose content obtained from 4
plants of each genotype (three homozygous transgenic lines of
Arabidopsis transgenic plants bearing the construct 35S:HaWRKY76
and WT control plants) evaluated both after 5 days of complete
submergence and one day after recovery. Different letters indicate
samples that are significantly different (p value<0.05).
[0050] FIG. 26 shows the average yield (at the end of the life
cycle) of 25-day-old plants of each genotype (three homozygous
lines of Arabidopsis transgenic plants bearing the construct
35S:HaWRKY76 and WT control plants) that were grown in standard
growing conditions, then completely submerged during 5 days, and
then recovered. Different letters indicate samples that are
significantly different (p value<0.05).
[0051] FIG. 27A shows rosette weight measured 6 and 7 days after
waterlogging was applied during a week in 25-day-old plants of each
genotype grown on poor soil conditions. FIG. 27B shows the membrane
stability of the respective plants, evaluated 5 and 7 days after
the start of waterlogging. FIG. 27C shows the stem length of the
respective plants after waterlogging. Different letters indicate
samples that are significantly different (p value<0.05).
[0052] FIGS. 28A and 28B show the average yield and the yield of
each plant at the end of the life cycle, respectively, after one
week of the waterlogging treatment that was applied to 25-day old
plants as described in FIG. 27. Different letters indicate samples
that are significantly different (p value<0.05).
[0053] FIG. 29 shows the average yield per genotype (three
homozygous transgenic lines of Arabidopsis transgenic plants
bearing the construct 35S:Ha WRKY76 and WT control plants) that
were subjected to a waterlogging treatment during one week after
being grown in standard soil. Different letters indicate samples
that are significantly different (p value<0.05).
[0054] FIG. 30 shows the number of rosette leaves exhibited by
plants belonging to three homozygous transgenic lines of
Arabidopsis transgenic plants bearing the construct 35S:Ha WRKY76
and WT control plants that were grown in standard conditions.
Different letters indicate samples that are significantly different
(p value<0.05).
[0055] FIG. 31 shows the stem lengths measured during the life
cycle of plants belonging to three homozygous transgenic lines of
Arabidopsis transgenic plants bearing the construct 35S:HaWRKY76
and WT control plants. Different letters indicate samples that are
significantly different (p value<0.05).
[0056] FIG. 32 is an image of an EMSA assay performed with purified
recombinant HaWRKY76-GST. 12N is a double stranded oligonucleotide
exhibiting a random sequence whereas W-Box is a double stranded
oligonucelotide having the canonical W-box.
[0057] FIG. 33 is a schematic representation of three kinds of
assays with different water levels: a) hardly on the substrate, b)
1 cm above the rosette leaves, and c) 2-4 cm below the basal
portion of the main inflorescence.
[0058] FIGS. 34A-C show HaWRKY76 expression in sunflower. FIG. 34A
shows 5-day-old seedlings well irrigated (control panel) or exposed
to severe water stress (drought panel). FIG. 34B shows 15-day-old
plantlets (time 0) exposed to severe drought stress and FIG. 34C
shows submergence during 11 days and recovery (R). Transcript
levels of HaWRKY76 were quantified by RT-qPCR, normalized with
sunflower actin (ACTIN2 and ACTIN8), and thereafter with respect to
the value (FIG. 34A) measured in the cotyledon sample under control
conditions, (FIG. 34B) in the sample exposed to water stress during
2 days, or (FIG. 34C) in the beginning of the treatment, the three
arbitrarily assigned a value of one. Two independent experiments
were done and error bars correspond to standard deviations from
three biological replicas in each experiment. An ANOVA test was
performed, followed by a Fisher LSD post-hoc test. Different
numbers of asterisks indicate samples with significant differences
(P<0.05).
[0059] FIGS. 35A-D show HaWRKY76 transgenic plants exhibit equal or
higher yield than WT after water deficit or water excess
treatments. FIGS. 35A and 35B show seed production of transgenic
(W76-A, W76-B, W76-C) and WT plants subjected to mild water deficit
in the vegetative (FIG. 35A) or the reproductive (FIG. 35B) stage.
FIGS. 35C and 35D show seed production of transgenic (W76-A, W76-B,
W76-C) and WT plants in the reproductive stage subjected to
submergence (FIG. 35C) or waterlogging (FIG. 35D). Two or three
plants per pot were assayed. Three experiments were done and error
bars correspond to standard deviations from four biological
replicas in each experiment. ANOVA test was performed, followed by
a Fisher LSD post-hoc test. Asterisks indicate samples which are
significantly different from WT (P<0.05)
DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION
Definitions
[0060] Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation, and amino acid sequences are
written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range and include each integer within the defined range. Amino
acids may be referred to herein by either their commonly known
three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
[0061] The term "amplified" refers to the construction of multiple
copies of a nucleic acid sequence or multiple copies complementary
to the nucleic acid sequence using at least one of the nucleic acid
sequence as a template. Amplification systems include, but are not
limited to, polymerase chain reaction (PCR), ligase chain reaction
(LCR) system, nucleic acid sequence based amplification (NASBA),
Q-Beta Replicase systems, transcription-based amplification system
(TAS), and strand displacement amplification (SDA). See, e.g., D.
H. Persing et al., "Diagnostic Molecular Microbiology: Principles
and Applications," American Society for Microbiology, Washington
D.C. (1993).
[0062] The term "introduced" or "introducing" as used herein in the
context of inserting into a cell refers to the incorporation of a
nucleic acid into a target cell, such as a plant cell, such that
the nucleic acid may be incorporated into the genome of the cell
(e.g., chromosome, plasmid, plastid or mitochondrial DNA),
converted into an autonomous replicon, or transiently expressed
(e.g., transfected mRNA). In embodiments, introducing a nucleotide
sequence into a plant cell results in transformation of the plant
cell to cause stable or transient expression of the sequence.
[0063] The term "isolated" as used herein refers to material, such
as a nucleic acid or a protein, which is substantially or
essentially free of components that normally accompany or interact
with the material within its naturally occurring environment. The
isolated material may include a material not found with the
material in its natural environment, or if the material is in its
natural environment, the material has been synthetically (i.e.,
non-naturally) altered by deliberate human intervention to form a
composition and/or be found in a location in the cell (e.g., genome
or subcellular organelle) not native to the material found in that
environment. The alteration forming the synthetic material can be
performed on the material within or removed from its natural state.
For example, a naturally occurring nucleic acid becomes an isolated
nucleic acid if it is altered, by means of human intervention on
the cell from which it originates. Likewise, a naturally occurring
nucleic acid (e.g., a promoter) becomes isolated if it is
introduced by non-naturally occurring means to a locus of the
genome not native to that nucleic acid, as discussed further
below.
[0064] As used herein, the term "nucleic acid" (or
"polynucleotide") refers to a deoxyribonucleotide or a
ribonucleotide polymer, or analog thereof, that has the essential
nature of natural nucleotides in that it hybridizes, under
stringent hybridization conditions, to substantially the same
nucleotide sequence as naturally occurring nucleotides and/or
allows translation into the same amino acid(s) as the naturally
occurring nucleotide(s). A polynucleotide can be full-length or a
sub-sequence of a native or heterologous structural or regulatory
gene. Unless otherwise indicated herein, the term refers to a
specified sequence, as well as the complementary sequence thereof.
Thus, DNAs or RNAs with backbones modified for stability or for
other reasons, as well as DNAs and RNAs comprising unusual or
modified bases, are polynucleotides as the term is defined herein.
The term polynucleotide also encompasses chemically, enzymatically,
or metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of simple and complex
cells. The term "nucleic acid" (or "polynucleotide") may be used in
place of, inter alia, gene, cDNA, mRNA, or cRNA.
[0065] As used herein, the term "operably linked" refers to a
functional linkage between sequences, such as a promoter and a
second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the
second sequence. Generally, operably linked means that the nucleic
acid sequences being linked are contiguous and, where necessary to
join two protein coding regions, contiguous in the same reading
frame.
[0066] The term "plant" is used broadly herein to describe a plant
at any stage of development, to a part of a plant (e.g., plant
cell, plant cell culture, plant organ, plant seed, etc.), and to
progeny thereof. A "plant cell" is the structural and physiological
unit of the plant, comprising a protoplast and a cell wall. A plant
cell can be in the form of an isolated single cell or a cultured
cell, or can be part of a higher organized unit, such as plant
tissue, a plant organ, or a plant. Thus, a plant cell can be a
protoplast, a gamete-producing cell, or a cell or collection of
cells that can regenerate into a whole plant. As used herein, a
"seed" comprises multiple plant cells and is capable of
regenerating into a whole plant, and may therefore be considered a
plant cell. A plant tissue or organ can be a seed, protoplast,
callus, or any other group of plant cells that is organized into a
structural or functional unit. Parts of a plant that are
particularly useful in embodiments include harvestable parts and
parts used for propagation of progeny plants. A harvestable part of
a plant may include the flowers, pollen, seedlings, tubers, leaves,
stems, fruit, seeds, roots, and the like. Parts of the plant used
for propagation include, e.g., seeds, fruits, cuttings, seedlings,
tubers, rootstocks, and the like. The class of plants that may be
used is generally as broad as the class of higher plants amenable
to transformation techniques, including both monocotyledonous and
dicotyledonous plants.
[0067] The terms "polypeptide" and "protein" as used herein refer
to a polymer of amino acid residues. The terms encompass amino acid
polymers, in which one or more amino acid residue is an artificial
chemical analogue of a corresponding naturally occurring amino
acid, as well as to naturally occurring amino acid polymers. The
essential nature of such analogues of naturally occurring amino
acids is that, when incorporated into a protein, the protein is
specifically reactive to antibodies elicited to the same protein
but consisting entirely of naturally occurring amino acids. The
polypeptide group includes, but is not limited to, DNA binding
proteins, protein kinases, protein phosphatases, GTP-binding
proteins, and receptors.
[0068] As used herein, the term "promoter" refers to a region of
DNA that is upstream from the start of transcription and that is
involved in recognition and binding of RNA polymerase and other
proteins to initiate transcription. The promoter may be any
polynucleotide sequence that shows transcriptional activity in the
host (target) plant cells, plant parts, or plants.
[0069] As used herein, the term "recombinant" refers to a cell or
vector that has been modified by the introduction of a heterologous
nucleic acid or a cell that is derived from a cell so modified. For
example, recombinant cells express genes that are not found in
identical form within the native (non-recombinant) form of the cell
or express native genes that are otherwise abnormally expressed,
under-expressed, or not expressed at all as a result of deliberate
human intervention. The term does not encompass the alteration of
the cell or vector by naturally occurring events (e.g., spontaneous
mutation, or natural transformation, transduction, or
transposition).
[0070] As used herein, the term "recombinant expression cassette"
(or "expression cassette") refers to a nucleic acid construct that
is recombinantly or synthetically generated with a series of
specified nucleic acid elements, that permits transcription of a
particular nucleic acid in a target cell. The recombinant
expression cassette can be incorporated into a plasmid, chromosome,
mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
Typically, the recombinant expression cassette portion of an
expression vector includes a nucleic acid to be transcribed and a
promoter.
[0071] As used herein, the term "regulatory element" means a
nucleotide sequence that, when operatively linked to a coding
region of a gene, effects transcription of the coding region such
that a ribonucleic acid (RNA) molecule is transcribed from the
coding region. Regulatory elements include promoters, enhancers,
silencers, 3'-untranslated or 5'-untranslated sequences of
transcribed sequences, e.g., a poly-A signal sequence or other
protein or RNA stabilizing element, or other gene expression
control elements known to regulate gene expression or the amount of
expression of a gene product.
[0072] The terms "residue", "amino acid residue", and "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide. The amino
acid may be a naturally occurring amino acid and, unless otherwise
limited, may encompass non-natural analogs of natural amino acids
that can function in a similar manner as naturally occurring amino
acids.
[0073] As used herein, "sequence identity" in the context of two
polynucleotide or polypeptide sequences refers to the residues in
the two sequences that are the same when aligned for maximum
correspondence over a comparison window of a contiguous and
specified segment of a polynucleotide sequence. When percentage of
sequence identity is used in reference to proteins, it is
recognized that residue positions that are not identical often
differ by conservative amino acid substitutions. Where sequences
differ in conservative substitutions, the percent sequence identity
may be adjusted upwards to correct for the conservative nature of
the substitution. Sequences differing by such conservative
mutations are said to have "sequence similarity." Methods for
making this adjustment are well known to persons skilled in the
art. The "percentage" of sequence identity means the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may include additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0074] As used herein, the term "transgenic plant" refers to a
plant that includes within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome of the transgenic plant, such
that the polynucleotide is passed on to successive generations. The
term "transgenic" is used herein to describe any cell, cell line,
callus, tissue, plant part or plant (also referred to herein as a
"target cell" or "host cell") the genotype of which has been
altered by the presence of a heterologous nucleic acid, and
includes transgenic plants that have been initially altered, as
well as those created by sexual crosses or asexual propagation from
the initial transgenic plants. The term "transgenic" as used herein
does not encompass the alteration of the genome by naturally
occurring events, such as random cross-fertilization or spontaneous
mutation.
[0075] As used herein, the term "vector" refers to a nucleic acid
used in the transfection of a target cell and into which can be
inserted a polynucleotide. Vectors are often replicons. Expression
vectors permit transcription of a nucleic acid inserted
therein.
[0076] As used herein, the term "wild-type" (or "WT") refers to a
cell or plant that has not been genetically modified to knock out
or over-express polypeptides according to embodiments of the
present disclosure. Wild-type cells or plants may be used as
controls to compare levels of expression and the extent and nature
of trait modification in genetically modified (i.e., transgenic)
cells or plants in which polypeptide expression is altered or
ectopically expressed by, for example, knocking out or
over-expressing a gene.
DETAILED DESCRIPTION OF EMBODIMENTS
[0077] The present invention will now be further described. In the
following passages, different aspects of the invention are defined
in more detail. Each aspect so defined may be combined with any
other aspect or aspects unless clearly indicated to the contrary.
In particular, any feature indicated as being preferred or
advantageous may be combined with any other feature or features
indicated as being preferred or advantageous. The practice of the
present invention will employ, unless otherwise indicated,
conventional techniques of botany, microbiology, tissue culture,
molecular biology, chemistry, biochemistry and recombinant DNA
technology, bioinformatics which are within the skill of the art.
Such techniques are explained fully in the literature.
[0078] External stress factors, from both biological and abiotic
origins, affect the levels of specific proteins by transcriptional
and/or post-transcriptional regulation. The ability of a given
species to survive various stress conditions is intimately related
to a series of molecular responses involving activation and
repression of certain genes. The stress tolerance of a species
appears to be controlled primarily at the transcriptional level,
depending on the transcription factor activity.
[0079] As in other species, the primary players regulating gene
expression in sunflowers are transcription factors and genomic
regulatory regions (as well as small RNAs, including miRNAs).
Transcription factors are proteins that are able to recognize and
bind specific DNA sequences present in the regulatory regions of
their target genes, and modulate their transcription. It is known
that transcription factors have a modular structure and exhibit at
least two types of domains: a DNA binding domain; and a
protein-protein interaction domain, which mediates (directly or
indirectly) the activation or repression of transcription. See
Brivanlou and Darnell, "Signal transduction and the control of gene
expression," Science 295:813-818 (2002). Additionally, transgenic
plants comprising isolated polynucleotides encoding transcription
factors may also modify expression of endogenous genes,
polynucleotides, and proteins. See Peng et al., Genes and
Development 11:3194-3205 (1997), and Peng et al., Nature
400:256-261 (1999).
[0080] Within a single plant species, gene duplication may cause
two copies of a particular gene, giving rise to two or more genes
with similar sequences and often similar functions, known as
paralogs. A paralog is therefore a similar gene formed by
duplication within the same species. Paralogs typically cluster
together or in the same clade (i.e., a group of similar genes) when
a gene family phylogeny is analyzed using programs known to persons
skilled in the art. For example, a clade of very similar
transcription factors of the same species typically will share a
common function (e.g., flowering time, drought tolerance, etc.).
Analysis of groups of similar genes having a similar function that
fall within one clade can yield sub-sequences that are particular
to that clade. These sub-sequences, known as "consensus sequences,"
can be used not only to define the sequences within each clade, but
also to define the functions of these genes; genes within a clade
may contain paralogous sequences, or orthologous sequences that
share the same function. See, e.g., Mount, "Bioinformatics:
Sequence and Genome Analysis," Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., p. 543 (2001).
[0081] Transcription factor gene sequences are conserved across
diverse eukaryotic species lines. Plants are no exception to this
observation; diverse plant species possess transcription factors
that have similar sequences and functions. Speciation for the
production of new species from a parental species gives rise to two
or more genes with similar sequence and similar function. These
genes, or "orthologs," often have an identical function within
their host plants and are often interchangeable between species
without losing function. Because plants have common ancestors, many
genes in any plant species will have a corresponding orthologous
gene in another plant species. Once a phylogenic tree for a gene
family of one species has been construed using a program, such as
CLUSTAL, potential orthologous sequences can be placed into the
phylogenetic tree and their relationship to genes from the species
of interest can be determined. Orthologous sequences can also be
identified by a reciprocal BLAST strategy. Once an orthologous
sequence has been identified, the function of the ortholog can be
deduced from the identified function of the reference sequence. By
using a phylogenetic analysis, persons skilled in the art would be
able to predict similar functions conferred by closely-related
polypeptides. An orthologous sequence of a plant, including plants
specifically mentioned herein, can have at least 70%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to the Ha nucleic
acid or protein described herein. The invention also includes
embodiments that relate to orthologous sequences.
[0082] Although approximately 2000 plant transcription factors have
been identified in plants in silico and classified in families and
sub-families according to similarities in the respective binding
domains, gene structures, functions and other structural features,
only a small percentage of those transcription factors has been
functionally characterized (even in model plants). The WRKY
transcription factors of Arabidopsis are an example of one such
classified family, which has also been functionally characterized
as related to abiotic and biotic stress responses. See, e.g.,
Giacomelli et al. (2010) and Giacomelli et al. (2012).
Specifically, the distinct transcription factors of the WRKY family
in Arabidopsis and numerous closely-related sequences from dicots
and monocots have been shown to confer increased water deprivation
tolerance.
[0083] The WRKY transcription factors are primarily characterized
by the presence of a 60 amino acid conserved region containing the
four WRKY amino acids and a zinc-finger-like motif, which together
form the WRKY domain. These proteins are unique to plants and have
been classified in Arabidopsis into three main groups (I, II, and
III) on the basis of both the number of WRKY domains and the
pattern of the zinc-finger-like motif, with the second group being
further classified in five subgroups (IIa, IIb, IIc, IId, and IIe).
See Eulgem and Somssich, "Networks of WRKY transcription factors in
defense signaling," Curr. Opin. Plant Biol. 10:366-371 (2007); and
Rushton et al., "WRKY transcription factors," Trends in Plant Sci.,
15:247-258 (2010).
[0084] In a study to identify and characterize WRKY transcription
in the sunflower (belonging to the Asteraceae family) using in
silico approaches and gene expression surveys, a bioinformatics
analysis of EST databases estimated the existence of 97 sunflower
WRKY members. See Giacomelli et al., "Expression analyses indicate
the involvement of sunflower WRKY transcription factors in stress
responses, and phylogenetic reconstruction reveal the existence of
a novel clade in the Asteraceae," Plant Science 178:398-410 (2010).
Phylogenetic trees constructed with WRKY domains resolved the same
seven groups assigned to Arabidopsis, as well as a novel clade
diverging with the IId subgroup as described above, with traits
apparently specific to Asteraceae. The 2010 study referenced above
indicated that the WRKY family has undergone a particular
diversification, which could be the source of specific new
functions. A partial sequence of HaWRKY76, a member of this
Asteraceae specific clade, was also reported in the study.
[0085] A schematic representation of the deduced proteins with a
WKKY motif corresponding to EST-clusters from Helianthus spp. and
Lactuca spp. is provided in Giacomelli et al. (2010), which shows
the putative conserved primary structural features of the
Helianthus/Lactuca WKKY(WKKYGEK, SEQ ID NO:7) motif encoding
proteins. All of the expressed sequence tags (ESTs) identified as
proteins having WKKY motif-encoding sequences (52) were obtained
from GenBank, and the EST-clusters resulting from the assembly (18)
were translated. In Giacomelli's schematic representation,
conserved primary structural features identified by using MEME
(Bailey and Elkan (1994)) are identified with grey boxes and
labels. Box A shows the N-terminal consensus sequence, box H shows
the HARF motif, box C shows the calmodulin-binding protein, box Z
shows the zinc cluster, and box P shows the proline-rich motif. An
alignment was performed using MAFFT (Lopez R. (1997)) and two
regions from it are shown in detail below the scheme described
above. The first region shown is the serine-rich region, which has
divergences in five identified clusters. The second region shows is
the WKKY domain.
[0086] The WKKY clade proteins present structural differences
relative to members of the WRKY family. Specifically, the WKKY
proteins contain motifs unique to the IId WRKY members, the C, the
HARF and a zinc cluster upstream of the WKKY domain (not the
zinc-finger-like motif proper of the WRKY domain). Moreover, they
exhibit an additional remarkable feature: the presence of two
substitutions in the main region of the WRKYGQK (SEQ ID NO:8)
domain that result in a WKKYGEK motif (SEQ ID NO:7). Other
signatures that have not been reported in other WRKY proteins, such
as a conserved N-terminal region, a putative serine-threonine
kinase domain, and proline-rich region (P), were also clearly
identified.
[0087] Since WKKY proteins share conserved structures with IId WRKY
group members, it is possible that they have some properties in
common. For example, the expression of members of the IId WRKY
group is induced by pathogen infection and SA. Additionally, EMSA
assays performed with AtWRKY11 showed that the substitution of RK
by KR in the WRKY domain avoided the interaction with the W-box.
See Ciolkowski et al., "Studies on DNA-binding selectivity of WRKY
transcription factors lend structural clues into WERKY-domain
function," Plant Molecular Biology 68:81-92 (2008). In contrast,
EMSA assays performed with HaWRKY76 indicated that this protein
selected a different DNA sequence. Because WKKY proteins share
conserved structures with IId WRKY members, it is possible that the
proteins have in common the properties associated with these
structures. Similar to the expression of proteins belonging to the
IId WRKY group being induced by pathogen infection and SA
concomitantly with a calcium discharge, the zinc cluster could be
involved in determining the DNA affinity as it was demonstrated for
AtWRKY11. However, this Arabidopsis transcription factor presented
a reduced affinity for a W-box when a single D to E amino acid was
substituted within the N-terminal region to the WRKY domain.
Because sunflower proteins with a WKKY motif do not have a D within
the zinc cluster, different amino acids could be determining the
affinity of these proteins for their target sequences.
[0088] The expression of sunflower WRKY genes is regulated by
hormones, abiotic and biotic factors, and wounding. The published
study of Giacomelli et al. (2010) includes a comparative diagram of
selected expression profiles of sunflower WRKY genes after
three-hour treatments with hormones or factors associated with
stress, i.e., the hormones CK (100 .mu.M 6-benzylaminopurine), GA
(100 .mu.M gibberellin A3), AUX (100 .mu.M indole-3-acetic acid),
ACC (30 .mu.M aminocyclopropane-carboxylic acid), JA (200 .mu.M
MeJA), and SA (1 mM), the wounding damage (WO), MAN 350 mM
D-mannitol), SALT (150 mM NaCl) and the Pseudomonas syringae DC3000
spraying (PS). The transcript levels were measured by quantitative
RT-PCR and standard errors were calculated from three biological
replicates in which actin transcripts (HaACTIN) were used as
internal controls.
[0089] The presence of the exclusive stretches found only in
members of the WKKY clade suggests that they could be the basis of
a neo-functionality. In this sense, the serine-rich sequence found
by the ELM program could be a target of a serine-threonine kinase.
PPLP motifs are proline-rich sequences present in ligands
interacting with WW domains. Notably, proline residues in the PPLP
motif identified in sunflower WKKYs are quite equally distributed
as in FY PPLP [PxP(x).sub.7PPLP], but the spacer sequences are
completely different.
[0090] Besides the 2010 publication and the subsequent Ph.D. Thesis
of Jorge Giacomelli (2011), no additional information regarding any
HaWRKY76 sequences (including the HaWRKY76 sequence of Giacomelli
(2010)) have been reported, and no functional analyses have been
conducted.
[0091] Using techniques described herein, the inventors have now
cloned the complete HaWRKY76 under the control of a 35S CaMV
promoter. The HaWRKY76 nucleotide sequence and polypeptide
(protein) sequence below correspond to SEQ ID NO: 1 and SEQ ID NO:
2, respectively. The HaWRKY76 transcription factor has a WKK motif
because there is a change of R to K in the WRKY domain. Thus,
HaWRKY76 is also referred to as a WKKY transcription factor
herein.
TABLE-US-00001 HaWRKY76 nucleotide sequence of SEQ ID NO: 1 (cDNA)
atggcggttgatttcgtcggaattcaatctaccgatcatcttctaa
accgcatgttccagttattaagtcacgatttaaacgtttcgtcaac
ctacacgcacgcggtttctgctttcaaacgcaccggtcacgcacgg
ttccgccgtggaccgtcgtctaccaccggagacactaacggacctt
caacttatcacattcggaaggtaaatcacgagatacgacttcgttt
gtacaaaacgagtgtttttcaaacaaaccggtgacggagataacga
cgacgacgacgtcaacgagctcgtcgtctgtagtatcgtcttccac
cggtggaaacttagacggaagtgtttccaacggtaaacagttttct
tcgttaggtatagtagctccggcgccgacgttctcgtctagaaaac
caccgttaccgtcgacacaccggaaaaggtgcggcgctgatcgtcc
tgttgcttccgtacacggatccggaagcggttgccattgttgttcc
aagagaaggaaaaccggatctaaacgtgaaattagaagagttccga
ttaccggatctaaaattacaagcatacctgctgatgattactcatg
gaaaaagtacggcgagaagaagatcgacggttcactttatccacga
gtatattacaaatgtattaccggaaaaggatgtccggcgaggaagc
gcgtggagttaagcgccgacgattcgaagatgcttattgttactta
cgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatg
agtttgaccggtgtgtatggtgagccaaagtgaa Corresponding HaWRKY76
polypeptide (protein) sequence of SEQ ID NO: 2
MAVDFVGIQSTDHLLNRMFQLLSHDLNVSSTYTHAVSAFKRTGHAR
FRRGPSSTTGDTNGPSTSSHSEGKSRDTTSFVQNECFSNKPVTEIT
TTTTSTSSSSVVSSSTGGNLDGSVSNGKQFSSLGIVAPAPTFSSRK
PPLPSTHRKRCGADRPVASVHGSGSGCHCCSKRRKTGSKREIRRVP
ITGSKITSIPADDYSWKKYGEKKIDGSLYPRVYYKCITGKGCPARK
RVELSADDSKMLIVTYDGEHRHRDRHAPVPMSLTGVYGEPK
[0092] A sequence having similarity with the HaWRKY76 sequences
disclosed herein has been reported in the Helia database. The gene
and protein sequences of HaT131007971 correspond to SEQ ID NO: 3
and SEQ ID NO: 4, respectively. A comparison of the HaT131007971
polypeptide (protein) sequence (SEQ ID NO: 4) with the full-length
polypeptide (protein) sequence of HaWRKY76 (SEQ ID NO: 2) indicates
several differences, as shown in FIG. 1. A comparison of the
HaT131007971 polynucleotide sequence (SEQ ID NO: 3) with the
full-length polynucleotide sequence of HaWRKY76 (SEQ ID NO: 1)
indicates several differences, as shown in FIG. 2.
[0093] Another sequence having similarity with the HaWRKY76
sequences disclosed herein is HuCL13748C001, which has also been
reported in the Helia database. The gene and protein sequences of
the HuCL13748C001 correspond to SEQ ID NO: 5 and SEQ ID NO: 6,
respectively. A comparison of the polypeptide (protein) sequence of
HuCL13748C001 (SEQ ID NO: 6) with the full-length polypeptide
(protein) sequence of HaWRKY76 (SEQ ID NO: 2) indicates several
differences, as shown in FIG. 3. A comparison of the polynucleotide
sequence of HuCL13748C001 (SEQ ID NO: 5) and the full-length
polynucleotide sequence of HaWRKY76 (SEQ ID NO: 1) indicates
several differences, as shown in FIG. 4. These differences have not
yet been evaluated for functional roles, but some of them are not
conservative and/or located in putative functional domains.
[0094] It is generally understood that the up-regulation of a
certain gene by any abiotic stress factor does not mean that the
gene will confer tolerance to such stress if it is used as a
transgene. Moreover, the ability of a gene to confer tolerance to a
given stress factor does not imply that it will confer tolerance to
other stress factors. Many examples of genes conferring tolerance
to drought but not to, e.g., high temperatures, are described in
scientific literature and understood by persons skilled in the art.
In the same way, tolerance to low temperatures above 0.degree. C.
(chilling) is generally not concomitant with tolerance to low
temperatures below 0.degree. C. (freezing) since different
molecular mechanisms are playing a role in these responses.
[0095] In the same way, it is also generally understood that
tolerance to drought does not imply tolerance to submergence or
waterlogging because different signal transduction pathways are
triggered in these responses. Likewise, tolerance to drought or to
any other abiotic stress factor does not imply increased yield
under such stresses. While tolerance is usually evaluated and
reported as a percentage of survivors after a severe stress
treatment, the yield under such conditions is generally not
informed under moderate stress conditions. See e.g., Skirycz et
al., "Survival and growth of Arabidopsis plants given limited water
are not equal," Nature Biotechnology 29:212-214 (2011) (reporting
that 25 genes known to confer drought tolerance exhibited decreased
yield in standard conditions or under a moderate drought stress).
Such moderate stress is the most common and probable growing
condition encountered by plants in the field.
[0096] Thus, a combination of increased yield (or at least no
decrease in yield) and stress tolerance represents a very valuable
characteristic of technologies to improve crops. As described in
connection with the various embodiments and specific Examples
provided herein, HaWRKY76 unexpectedly offers this highly
advantageous combination of characteristics.
[0097] As indicated above, the complete HaWRKY76 was cloned under
the control of the 35S CaMV promoter. This construct was further
used to transform Arabidopsis plants to produce transgenic plants
exhibiting different expression levels of HaWRKY76. Plants produced
according to the methods described herein were analyzed both in
standard growth conditions and in growth conditions in which they
were subjected to abiotic stress factors.
[0098] The analysis revealed, among other things, that when grown
under standard conditions, 35S:HaWRKY76 transgenic plants have a
similar number of rosette leaves, life cycle duration and stem
length as the corresponding WT control plants. However, transgenic
plants bearing the construct 35S:HaWRKY76 were found to have longer
roots and larger rosettes than corresponding WT control plants.
Moreover, total protein and chlorophyll contents of the transgenic
plants were found to be proportional to the rosette weight (i.e.,
35S:HaWRKY76 plants produce more biomass and protein than control
plants). Furthermore, when subjected to water stress, transgenic
plants bearing the construct 35S:HaWRKY76 were found to be more
tolerant to drought than corresponding WT control plants, and
similar properties were observed when the transgenic plants were
stressed by submergence or waterlogging. Notably, transgenic plants
bearing the construct 35S:HaWRKY76 not only demonstrated improved
tolerance to the various moderate and severe stress conditions, but
also exhibited higher yields than the corresponding WT control
plants (yield evaluated as a measure of seed production).
[0099] The HaWRKY76 transcription factor polypeptides confer on
transgenic plants produced according to methods described herein
improved stress tolerance, as well as increased yield in standard
growing conditions. Specifically, as evidenced by the various
Examples and applicable Figures, the inventors unexpectedly
discovered that the HaWRKY76 sequences confer tolerance to drought,
submergence and waterlogging, while also increasing yield in
standard conditions.
[0100] It will be understood by persons skilled in the art that
embodiments of the invention also relate to, among other things,
the isolation and functional characterization of the HaWRKY76
nucleotide and amino acid sequences, transgenic plants transformed
with constructs comprising the HaWRKY76 polynucleotide sequences,
and methods of producing transgenic plants expressing the HaWRKY76
polypeptide sequences disclosed herein, wherein the transgenic
plants have improved stress tolerance and increased yield in
comparison to corresponding control plants, for example WT
plants.
[0101] WRKY76 Transcription Factor Polynucleotides
[0102] In embodiments of the aspects of the invention, the
polynucleotides described herein include nucleotide sequences that
encode WRKY76 transcription factors and transcription factor
homolog polypeptides and sequences complementary thereto, as well
as unique fragments of a coding sequence, or a sequence
complementary thereto. The polynucleotides may be, e.g., DNA or
RNA, such as mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic
DNA, oligonucleotides, etc. The polynucleotides are either
double-stranded or single-stranded and include either or both sense
(i.e., coding) sequences and antisense (i.e., non-coding,
complementary) sequences. The polynucleotides may include the
coding sequence of a transcription factor or transcription factor
homolog polypeptide, in isolation, in combination with additional
coding sequences, in combination with non-coding sequences (e.g.,
introns, regulatory elements such as promoters, enhancers,
terminators, and the like), and/or in a vector or host environment
in which the polynucleotide encoding a transcription factor or
transcription factor homolog polypeptide is an endogenous or
exogenous gene. WRKY76 transcription factors include the signature
motif WKKYGEK (SEQ ID NO:7). WRKY76 transcription factors also
include a conserved serine-threonine kinase domain and a
proline-rich region.
[0103] Representative polynucleotides of WRKY76 transcription
factors include the full-length polynucleotide sequence of SEQ ID
NO: 1, and functional variants or parts thereof which retain
biological function of the full-length polynucleotide sequence of
SEQ ID NO: 1 or SEQ ID NO:9, for example SEQ ID NO: 11. Preferably,
variants are substantially identical polynucleotides. Substantially
identical polynucleotides comprise nucleotide sequences that vary
from the full-length amino acid sequence of SEQ ID NO: 1 by one or
more modifications, including deletions, substitutions, or
additions, the net effect of which is retained biological function
of the WRKY76 polynucleotide. For example, substantially identical
polynucleotides comprise nucleotide sequences that vary from the
full-length amino acid sequence of SEQ ID NO: 1 by 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 substitutions, or additions. In some embodiments,
variants, such as substantially identical WRKY76 polynucleotides
may have at least 80% sequence identity with the full-length
nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:9 as described
herein, or by visual inspection. Preferably, the WRKY76
polynucleotides have at least 85% sequence identity, or at least
90% sequence identity, or at least 91% sequence identity, or at
least 92% sequence identity, or at least 93% sequence identity, or
at least 94% sequence identity, or at least 95% sequence identity,
or at least 96% sequence identity, or at least 97% sequence
identity, or at least 98% sequence identity, or at least 99%
sequence identity with the full-length nucleotide sequence of SEQ
ID NO: 1. For example, sequence identity is at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 8'7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100%. A variant within the scope of the
various aspects of the invention is shown in SEQ ID NO: 11 and the
corresponding polypeptide SEQ ID NO: 12.
[0104] In some embodiments of the various aspects of the invention,
the isolated polynucleotides of the invention contain at least one
of the following: (a) adenine as the nucleotide corresponding to
position 817 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO: 11; (b) cytosine as
the nucleotide corresponding to position 808 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO: 11; (c) cytosine as the nucleotide corresponding
to position 33 of the full-length nucleotide sequence of SEQ ID NO:
1 or a variant thereof, for example SEQ ID NO: 11; (d) cytosine as
the nucleotide corresponding to position 259 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO: 11; and (e) guanine as the nucleotide
corresponding to position 315 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO: 11. In particular, in one aspect, the invention relates to an
isolated polynucleotide having at least 80% sequence identity with
the full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO: 11, wherein the polynucleotide
contains at least one of: [0105] (a) adenine as the nucleotide
corresponding to position 817 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO: 11; [0106] (b) cytosine as the nucleotide corresponding to
position 808 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO: 11; [0107] (c)
cytosine as the nucleotide corresponding to position 33 of the
full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO: 11; [0108] (d) cytosine as the
nucleotide corresponding to position 259 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO: 11; and [0109] (e) guanine as the nucleotide
corresponding to position 315 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO: 11. Preferably, the isolated polynucleotide is cDNA.
[0110] Substantially identical polynucleotide sequences may be
polymorphic sequences, i.e., alternative sequences or alleles in a
population in which the allelic difference may be as small as one
base pair. Substantially identical polynucleotides may also
comprise mutagenized sequences, including sequences comprising
silent mutations. A mutation may comprise one or more residue
changes, a deletion of one or more residues, or an insertion of one
or more additional residues.
[0111] Representative polynucleotides according to some embodiments
of the various aspects of the invention include the polynucleotide
comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 9 and
substantially identical nucleotides, and polynucleotides that
encode the HaWRKY76 transcription factor polypeptide comprising or
consisting of SEQ ID NO: 2. Thus, in one embodiment, the isolated
polynucleotide comprises or consists of SEQ ID NO:1. In one
embodiment, the isolated polynucleotide comprises or consists of
SEQ ID NO:1.
[0112] Substantially identical polynucleotides according to
embodiments include polynucleotides that hybridize specifically to
or hybridize substantially to the full-length nucleotide sequence
of SEQ ID NO: 1, 11 or 9 under stringent conditions. In the context
of nucleic acid hybridization, two nucleotide sequences being
compared may be designated as a probe and a target. A probe is a
reference nucleic acid molecule, and a target is a test nucleic
acid molecule, often found within a heterogeneous population of
nucleic acid molecules. In this respect, a target sequence is
synonymous with a test sequence.
[0113] In some embodiments of the aspects of the invention, the
polynucleotides include primers and primer pairs that allow
specific amplification of the disclosed polynucleotides or of any
specific parts thereof, and probes that selectively or specifically
hybridize to nucleic acid molecules of the invention or to any part
thereof. Primers may also be used as probes and can be labeled with
a detectable marker, such as, for example, a radioisotope,
fluorescent compound, bioluminescent compound, a chemiluminescent
compound, metal chelator or enzyme. A particular nucleotide
sequence employed for hybridization studies or assays may include
probe sequences that are complementary to at least about 14-40
nucleotide sequence of a nucleic acid molecule described herein.
Probes may comprise 14-20 nucleotides, or even longer where
desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides
or up to the full length of SEQ ID NO: 1, 11 or SEQ ID NO: 9. Such
fragments may be readily prepared, for example by chemical
synthesis of the fragment, by application of nucleic acid
amplification technology, or by introducing selected sequences into
recombinant vectors for recombinant production.
[0114] Specific hybridization refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent conditions when that sequence is present in a
complex nucleic acid mixture (e.g., total cellular DNA or RNA).
Specific hybridization may accommodate mismatches between the probe
and the target sequence depending on the stringency of the
hybridization conditions.
[0115] Stringent hybridization conditions and stringent
hybridization wash conditions in the context of nucleic acid
hybridization experiments, such as Southern and Northern blot
analysis, are both sequence- and environment-dependent. Longer
sequences hybridize specifically at higher temperatures. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2,
Elsevier, New York, N.Y. (1993). Generally, stringent hybridization
and wash conditions are selected to be about 5.degree. C. below the
thermal melting point for the specific sequence at a defined ionic
strength and pH. Typically, under stringent conditions a probe will
hybridize specifically to its target sequence, but not to other
sequences.
[0116] The Tm is the temperature (under defined ionic strength and
pH) at which 50% of the target sequence hybridizes to a perfectly
matched probe. Stringent conditions are selected to be equal to the
Tm for a particular probe. An example of stringent hybridization
conditions for Southern or Northern Blot analysis of complementary
nucleic acids having more than about 100 complementary residues is
overnight hybridization in 50% formamide with 1 mg of heparin at
42.degree. C. An example of highly stringent conditions is 15
minutes in 0.1.times.SSC at 65.degree. C., whereas an example of
stringent wash conditions is 15 minutes in 0.2.times.SSC buffer at
65.degree. C. See Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989). Typically, a high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example of
medium stringency conditions for a duplex of more than about 100
nucleotides is 15 minutes in 1.times.SSC at 45.degree. C. An
example of low stringency for a duplex of more than about 100
nucleotides is 15 minutes in 4 to 6.times.SSC at 40.degree. C. For
short probes (e.g., about 10 to 50 nucleotides), stringent
conditions typically involve salt concentrations of less than about
1M Na.sup.+ ion, typically about 0.01 to 1M Na.sup.+ ion
concentration (or other salts) at pH 7.0-8.3, and the temperature
is typically at least about 30.degree. C. Stringent conditions may
also be achieved with the addition of destabilizing agents, such as
formamide. Additional variations of these conditions will be
readily apparent to those skilled in the art.
[0117] Stringency conditions may be selected such that an
oligonucleotide that is perfectly complementary to the coding
oligonucleotide hybridizes the coding oligonucleotide with at least
about 5 to 10 times higher signal to noise ratio than the ratio for
hybridization of the perfectly complementary oligonucleotide to a
nucleic acid encoding a transcription factor know in the art. It
may be desirable to select conditions for a particular assay such
that a higher signal to noise ratio (e.g., about 15.times. or more)
is obtained. Accordingly, a subject nucleic acid will hybridize to
a unique coding oligonucleotide with at least 2 times (2.times.) or
greater signal to noise ratio as compared to hybridization of the
coding oligonucleotide to a nucleic acid encoding a known
polypeptide. The particular signal will depend on the label used in
the relevant assay, e.g., a fluorescent label, a calorimetric
label, a radioactive label, or the like. Labeled hybridization or
PCR probes for detecting related polynucleotide sequences may be
produced by oligolabeling, nick translation, end-labeling, or PCR
amplification using a labeled nucleotide.
[0118] A further indication that two nucleotide sequences are
substantially identical is that proteins encoded by the
polynucleotides are substantially identical, share an overall
three-dimensional structure, or are biologically functional
equivalents. Nucleic acid molecules that do not hybridize to teach
other under stringent conditions are still substantially identical
if the corresponding proteins are substantially identical. This may
occur, for example, when two nucleotide sequences comprise
conservatively substituted variants as permitted by the genetic
code. Conservatively substituted variants refer to nucleotide
sequences having degenerate codon substitution wherein the third
position of one or more (or all) codons is/are substituted with
mixed-base and/or deoxyinosine residues. See Batzer et al., Nucleic
Acids Res, 19:5081 (1991), Ohtsuka et al., J. Biol. Chem.
260:2605-2608 (1991); and Rossolini et al., Mol. Cell. Probes
8:91-98 (1994).
[0119] Methods using manual alignment of sequences similar or
homologous to one or more polynucleotide sequences or one or more
polypeptides encoded by the polynucleotide sequences may be used to
identify regions of similarity and conserved domains. Such manual
methods are well known to persons skilled in the art and can
include, for example, comparisons of the tertiary structure between
a polypeptide sequence encoded by a polynucleotide that comprises a
known function with a polypeptide sequence encoded by a nucleotide
sequence that has a function not yet determined. Examples of
tertiary structure may include predicted alpha helices,
beta-sheets, amphipathic helices, leucine zipper motifs, zinc
finger motifs, proline-rich regions, cysteine repeat motifs, and
the like.
[0120] In embodiments of the aspects of the invention, the
polynucleotides may include polynucleotides encoding the WRKY76
transcription factor polypeptide (protein) of SEQ ID NO: 2 or a
variant thereof, such as a WRKY76 transcription factor polypeptide
derived from the full-length amino acid sequence of SEQ ID NO: 2
containing one or more substitutions, deletions and/or additions of
amino acid residues. The polynucleotides may therefore also include
polynucleotides encoding a functional variant WRKY76 polypeptide as
described herein, for example SEQ ID NO: 12.
[0121] Orthologs and paralogs of transcription factor polypeptides
described herein may be cloned according to conventional methods.
These may have at least 80%, 85%, 90 or 95% sequence identity to
HaWRKY76. For example, cDNAs can be cloned using mRNA from a plant
cell or tissue that expresses one of the transcription factor
polypeptides described herein. Appropriate mRNA sources may be
identified by interrogating Northern blots with probes designed
from amino acid sequences within the scope of the present
disclosure, after which a library is prepared from the mRNA
obtained from a positive cell or tissue. Transcription
factor-encoding cDNA is then isolated by, for example, PCR, using
primers designed from a transcription factor gene sequence
disclosed herein, or by probing with a partial or complete cDNA or
with one or more sets of degenerate probes based on sequences
disclosed herein. The cDNA library may be used to transform plant
cells, as discussed further below, and expression of the cDNAs of
interest is detected using, for example, methods known or described
herein, such as microarrays, Northern blots, quantitative PCR, or
any other technique for monitoring changes in expression. Genomic
clones may also be isolated using similar techniques.
[0122] In embodiments of the aspects of the invention, the
polynucleotides may be cloned, synthesized, altered, mutagenized,
or combinations thereof. A nucleic acid can be isolated using
standard molecular biology techniques and the sequence information
provided herein. Standard recombinant DNA and molecular cloning
techniques used to isolate nucleic acids are known to persons
skilled in the art. In embodiments, a nucleic acid molecule can be
amplified using cDNA or, alternatively, genomic DNA, as a template
and appropriate oligonucleotide primers according to standard PCT
amplification techniques. The nucleic acid molecule so amplified
can be cloned into an appropriate vector and characterized by DNA
sequence analysis.
[0123] WRKY76 Transcription Factor Polypeptides (Proteins)
[0124] Embodiments according to the various aspects of the
invention also relate to isolated WRKY76 transcription factor
polypeptides. The term polypeptides (proteins) refers to compounds
made up of a single chain of amino acids joined by peptide
bonds.
[0125] Representative polypeptides according to embodiments of the
various aspects of the invention include the full-length amino acid
sequence of SEQ ID NO: 2, and variants thereof, including
substantially identical polypeptides. One variant is shown in SEQ
ID NO: 12 Substantially identical polypeptides have amino acid
sequences that vary from the full-length amino acid sequence of SEQ
ID NO: 2 by one or more deletions, substitutions, or additions, the
net of which is retained biological function of the WRKY76
polypeptide. In embodiments, the WRKY76 polypeptides have at least
80% sequence identity with the full-length amino acid sequence of
SEQ ID NO: 2 as described herein, or by visual inspection. In
embodiments, the WRKY76 polypeptides have at least 85% sequence
identity, or at least 90% sequence identity, or at least 91%
sequence identity, or at least 92% sequence identity, or at least
93% sequence identity, or at least 94% sequence identity, or at
least 95% sequence identity, or at least 96% sequence identity, or
at least 97% sequence identity, or at least 98% sequence identity,
or at least 99% sequence identity with the full-length amino acid
sequence of SEQ ID NO: 2.
[0126] In some embodiments, substantially identical polypeptides
contain at least one of the following: (a) proline as the amino
acid corresponding to position 270 of the full-length amino acid
sequence of SEQ ID NO: 2 or a variant thereof, for example SEQ ID
NO: 12; (b) proline as the amino acid corresponding to position 260
of the full-length amino acid sequence of SEQ ID NO: 2 or a variant
thereof, for example SEQ ID NO: 12; (c) proline as the amino acid
corresponding to position 87 of the full-length amino acid sequence
of SEQ ID NO: 2 or a variant thereof, for example SEQ ID NO: 12;
(d) serine as the amino acid corresponding to position 123 of the
full-length amino acid sequence of SEQ ID NO: 2 or a variant
thereof, for example SEQ ID NO: 12; (e) leucine as the amino acid
corresponding to position 14 of the full-length amino acid sequence
of SEQ ID NO: 2 or a variant thereof, for example SEQ ID NO: 12;
(f) leucine as the amino acid corresponding to position 22 of the
full-length amino acid sequence of SEQ ID NO: 2 or a variant
thereof, for example SEQ ID NO: 12; and (g) serine as the amino
acid corresponding to position 23 of the full-length amino acid
sequence of SEQ ID NO: 2 or a variant thereof, for example SEQ ID
NO: 12. In particular, in one aspect, the invention relates to an
isolated polypeptide comprising a sequence having at least 80%
sequence identity with the full-length amino acid sequence of SEQ
ID NO: 2, the polypeptide sequence containing at least one of:
[0127] (a) proline as the amino acid corresponding to position 270
of the full-length amino acid sequence of SEQ ID NO: 2 or a variant
thereof, for example SEQ ID NO: 12; [0128] (b) proline as the amino
acid corresponding to position 87 of the full-length amino acid
sequence of SEQ ID NO: 2 or a variant thereof, for example SEQ ID
NO: 12; [0129] (c) proline as the amino acid corresponding to
position 260 of the full-length amino acid sequence of SEQ ID NO: 2
or a variant thereof, for example SEQ ID NO: 12; [0130] (d) serine
as the amino acid corresponding to position 123 of the full-length
amino acid sequence of SEQ ID NO: 2 or a variant thereof, for
example SEQ ID NO: 12; [0131] (e) leucine as the amino acid
corresponding to position 14 of the full-length amino acid sequence
of SEQ ID NO: 2 or a variant thereof, for example SEQ ID NO: 12;
[0132] (f) leucine as the amino acid corresponding to position 22
of the full-length amino acid sequence of SEQ ID NO: 2 or a variant
thereof, for example SEQ ID NO: 12; and [0133] (g) serine as the
amino acid corresponding to position 23 of the full-length amino
acid sequence of SEQ ID NO: 2 or a variant thereof, for example SEQ
ID NO: 12.
[0134] Substantially identical sequences according to the
embodiments of the various aspects of the invention may be
polymorphic sequences, i.e., alternative sequences or alleles in a
population in which the allelic difference may be as small as one
base pair. Substantially identical polynucleotides may also
comprise mutagenized sequences, including sequences comprising
silent mutations. A mutation may comprise one or more residue
changes, a deletion of one or more residues, or an insertion of one
or more additional residues. In some embodiments, polypeptide
variants can be functional fragments of the WRKY76 transcription
factor polypeptide of SEQ ID NO: 2. Functional polypeptides may
include amino acid sequences that are longer than the sequences
described herein. For example, one or more amino acids may be added
to the N-terminus or C-terminus of a polypeptide. Such additional
amino acids may be employed in a variety of applications, including
(but not limited to) purification applications. Methods of
preparing elongated polypeptides are known to persons skilled in
the art. In one embodiment, the isolated polypeptide comprises or
consists of SEQ ID NO:2 or 12.
[0135] WRKY76 transcription factor polypeptides described herein
and according to the various aspects of the invention may comprise
naturally occurring amino acids, synthetic amino acids, genetically
encoded amino acids, non-genetically encoded amino acids, and
combinations thereof.
[0136] WRKY76 polypeptides described herein and according to the
various aspects of the invention may also include polypeptides
comprising amino acids that are conservatively substituted variants
of the full-length amino acid sequence of SEQ ID NO: 2. A
conservatively substituted variant refers to a polypeptide
comprising an amino acid in which one or more residues have been
conservatively substituted with a functionally similar residue.
Examples of conservative substitutions include the substitution of
one non-polar (hydrophobic) residue (e.g., isoleucine, valine,
leucine, or methionine) for another, such as between arginine and
lysine, between glutamine and asparagine, between glycine and
serine; the substitution of one basic residue (e.g., lysine,
arginine, or histidine) for another; or the substitution of one
acidic residue (e.g., aspartic acid or glutamic acid) for
another.
[0137] Isolated polypeptides according to embodiments may be
purified and characterized using a variety of standard techniques
that are known to persons skilled in the art. See Schroder et al.,
The Peptides, Academic Press, New York, N.Y. (1965).
[0138] Regulatory Elements
[0139] The invention also relates to a vector or nucleic acid
construct comprising a polynucleotide as described above. In one
embodiment, said isolated polynucleotide comprises or consists of
SEQ ID NO: 1 or 11. In another aspect, the invention relates to a
recombinant expression cassette comprising a polynucleotide as
described above, wherein the polynucleotide is operably linked to a
promoter. The polynucleotide may be in a sense or antisense
orientation. In one embodiment, said isolated polynucleotide
comprises or consists of SEQ ID NO: 1. In another aspect, the
invention relates to recombinant expression cassette comprising an
isolated polynucleotide operably linked to a promoter, wherein the
polynucleotide is a member selected from the group consisting of:
[0140] (a) a polynucleotide that encodes the polypeptide of SEQ ID
NO: 2 or 12; and [0141] (b) the polynucleotide of SEQ ID NO: 1 or a
variant thereof, for example SEQ ID NO: 11.
[0142] A regulatory element generally can increase or decrease the
amount of transcription of a nucleotide sequence operatively linked
to the element with respect to the level at which the nucleotide
sequence would be transcribed absent the regulatory element.
[0143] In some embodiments of the aspects of the invention,
stress-regulated regulatory elements, which regulate expression of
an operatively linked nucleotide sequence in a plant in response to
a stress condition, are also provided. The plant stress-regulated
regulatory elements may be isolated from a polynucleotide sequence
of a plant stress-regulated gene. Specifically, the plant
stress-regulated regulatory elements may be isolated from a
polynucleotide sequence of the WRKY76 gene comprising the
nucleotide sequence of SEQ ID NO: 1, or a variant thereof or
comprising a nucleotide sequence that is functionally equivalent to
the full-length nucleotide sequence of SEQ ID NO: 1, for example
SEQ ID NO: 11. Thus, a WRKY76 promoter, for example the HaWRKY76
promoter, may be used. This can be selected from SEQ ID NO: 10 or a
sequence with at least 80%, 85%, 90%, or 95% sequence identity with
SEQ ID NO: 10.
[0144] Methods for identifying and isolating a stress-regulated
regulatory element from the polynucleotides, or genomic DNA clones
corresponding thereto, are known to persons skilled in the art. For
example, methods of making deletion constructs or linker-scanner
constructs can be used to identify nucleotide sequences that are
responsive to a stress condition. Generally, such constructs
include a reporter gene operatively linked to the sequence to be
examined for regulatory activity. By performing such assays, a
plant stress-regulated regulatory element can be defined within a
sequence of about 500 nucleotides or fewer, generally at least
about 200 nucleotides or fewer, or about 50 to 100 nucleotides.
Preferably, the minimal (core) sequence required for regulating a
stress response of a plant is identified. The nucleotide sequences
of the genes of a cluster can also be examined using a homology
search engine to identify sequences of conserved identity,
particularly in the nucleotide sequence upstream of the
transcription start site.
[0145] Regulatory elements, as described and defined herein, may be
isolated from a naturally occurring genomic DNA sequence or can be
synthetic (e.g., a synthetic promoter). The regulatory elements can
be constitutively expressed so as to maintain gene expression at a
relative level of activity (basal level), or can be regulated.
Constitutively expressed regulatory elements can be expressed in
any cell type, or can be tissue specific (expressed only in
particular cell types), or phase specific (expressed only during
particular developmental or growth stages of a plant cell).
Regulatory elements (e.g., a tissue specific, phase specific, or
inducible regulatory element) useful in constructing a recombinant
polynucleotide or in practicing methods described herein include
regulatory elements that are found in a plant genome. In some
embodiments, the regulatory elements may be from an organism other
than a plant, such as a plant or animal virus, or an animal or
other multicellular organism.
[0146] In some embodiments, a regulatory element that is a promoter
element is provided. Particularly useful promoters include, but are
not limited to, constitutive, inducible, temporally regulated,
developmentally regulated, spatially-regulated, chemically
regulated, stress-responsive, tissue-specific, viral and synthetic
promoters. Promoter sequences are generally understood to be strong
or weak. A strong promoter provides for a high level of gene
expression, whereas a weak promoter provides for a low level of
gene expression. An inducible promoter is a promoter that allows
gene expression to be turned on and off in response to an
exogenously added agent, or to an environmental or developmental
stimulus. An isolated promoter sequence that is a strong promoter
for heterologous nucleic acid is typically advantageous because it
provides for a sufficient level of gene expression to allow for
easy detection and selection of transformed cells, while providing
a high level of gene expression when desired.
[0147] Several domains within a plant promoter region are necessary
for the full function of the promoter. The first of these domains
within the promoter region lies immediately upstream of the
structural gene and forms the "core promoter region" containing
consensus sequences, normally 70 base pairs immediately upstream of
the gene. The core promoter region represents a transcription
initiation sequence that defines the transcription start point for
the structural gene. The presence of the core promoter region
defines a sequence as being a promoter; that is, if the region is
absent, the promoter is non-functional. The core promoter region on
its own is, however, insufficient to provide full promoter
activity. A series of regulatory sequences upstream of the core
constitute the remainder of the promoter. These regulatory
sequences determine expression levels, the spatial and temporal
patterns of expression and, for the specific subset of promoters,
the expression level under inductive conditions (e.g., light,
temperature, chemicals, hormones).
[0148] To define a minimal promoter region, a DNA segment
representing the promoter region is removed from the 5'-region of
the gene of interest and operably linked to the coding sequence of
a marker (reporter) gene by recombinant DNA techniques known to
persons skilled in the art. The reporter gene is operably linked
downstream of the promoter, so that transcripts initiating at the
promoter proceed through the reporter gene. Reporter genes
generally encode proteins that are easily measured. The construct
containing the reporter gene under the control of the promoter is
then introduced into an appropriate plant cell by transfection
techniques known to persons skilled in the art. The level of enzyme
activity corresponds to the amount of enzyme produced, which, in
turn, reveals the level of expression from the promoter of
interest. This level of expression can be compared to that achieved
using other promoters to determine the relative strength of the
promoter under study. To ensure that the expression level is due to
the promoter, rather than the stability of the mRNA, the level of
the reporter mRNA can be measured directly (e.g., by Northern blot
analysis).
[0149] Once enzyme activity is detected, mutational and/or
deletional analyses may be performed to determine the minimal
region and/or sequences required to initiate transcription.
Sequences may be deleted at the 5'-end of the promoter region
and/or at the 3'-end of the promoter region, and nucleotide
substitutions may be introduced. These constructs may then be
introduced into cells and their activity determined.
[0150] The promoter selection depends on the temporal and spatial
requirements for expression, as well as on the target species. In
some embodiments, expression in multiple tissues may be desirable,
while in others, tissue-specific (e.g., leaf-specific,
seed-specific, petal-specific, anther-specific, or pith-specific)
expression is desirable. Although promoters from dicotyledons have
been shown to be operational in monocotyledons, and vice versa,
dicotyledonous promoters are ideally selected for expression in
dicotyledons, and monocotyledonous promoters are ideally selected
for expression in monocotyledons. There is no restriction as to the
origin or source of the promoter selected; it is sufficient that
the selected promoter is operational in driving the expression of a
nucleotide sequence described herein in the particular cell. That
is, the promoter used in embodiments of the present disclosure may
be any nucleotide sequence that shows transcriptional activity in
the target (host) plant (cell, seed, etc.).
[0151] Accordingly, in embodiments, the promoter may be native or
analogous, or foreign or heterologous, to the plant host and/or to
the DNA sequence disclosed herein. Where the promoter is native or
endogenous to the plant host, it is intended that the promoter is
found in the native plant into which the promoter is introduced.
Where the promoter is foreign or heterologous to the DNA sequence
disclosed herein, the promoter is not the native or naturally
occurring promoter for the operably linked DNA sequence disclosed
herein. The promoter selected in embodiments may be "inducible" or
"constitutive." An inducible promoter is a promoter that is under
environmental control, whereas a constitutive promoter is a
promoter that is active under most environmental conditions.
Moreover, the promoter may be naturally-occurring, composed of
portions of various naturally-occurring promoters, or partially or
totally synthetic. Guidance for the design of promoters is provided
by studies of promoter structure in Harley et al., Nucleic Acids
Res. 15:2343-61 (1987). Additionally, the location of the promoter
relative to the transcription start position may be optimized. See
e.g., Roberts et al., Proc. Natl. Acad. Sci. 76:760-764, USA
(1979).
[0152] For example, suitable constitutive promoters for use in
plants according to the present disclosure may include promoters
from plant viruses, such as the peanut chlorotic streak
caulimovirus (PC1SV) promoter, the 35S promoter from cauliflower
mosaic virus (CaMV), promoters of Chlorella virus methyltransferase
genes, the full-length transcript promoter from figwort mosaic
virus (FMV); the promoters from such genes as rice actin,
ubiquitin, pEMU, MAS, maize H4 histone, Brassica napus ALS4; and
promoters of various Agrobacterium genes. See e.g., Odell et al.,
Nature 313:810-812 (1985); McElroy et al., Plant Cell 2:163-171
(1990); Christensen et al., Plant Mol. Biol. 12:619-632 (1989);
Christensen et al., Plant Mol. Biol. 18:675-689 (1992); Last et
al., Theor. Appl. Genet. 81:581-588 (1991); Velten et al., EMBO J.
3:2723-27310 (1984); Lepetit et al., Mol. Gen. Genet. 231:276-285
(1992); and U.S. Pat. Nos. 4,771,002; 5,102,796; 5,182,200;
5,428,147; 5,850,019; 5,563,328; 5,378,619.
[0153] Suitable inducible promoters for use in plants according to
the present disclosure may include, for example, the promoter from
the ACE1 system that responds to copper, the promoter of the maize
In2 gene that responds to benzenesulfonamide herbicide safeners,
and the promoter of the Tet repressor from Tn10. See Mett et al.,
Proc. Natl. Acad. Sci., 90:4567-4571, USA (1993); Hershey et al.,
Mol. Gen. Genet. 227:229-237 (1991); and Gatz et al., Mol. Gen.
Genet. 243:32-38 (1994). Another inducible promoter that may be
used in plants described herein is one that responds to an
inducting agent to which plants do not normally respond. An
inducible promoter of this type may be the inducible promoter from
a steroid hormone gene, the transcriptional activity of which is
induced by glucocorticosteroid hormone, or the recent application
of a chimeric transcription activator, SVE, for use in an estrogen
receptor-based inducible plant expression system activated by
estradiol. See Schena et al., Proc. Natl. Acad. Sci. 88:104-21
(1991); Zuo et al., Plant J. 24:265-273 (2000). Other inducible
promoters suitable for use in embodiments may be selected from
promoters described in EP 332104, PCT International Publication
Nos. WO93/21334 and WO 97/06269. Promoters composed of portions of
other promoters and partially or totally synthetic promoters may
also be used. See e.g., Ni et al., Plant J. 7:661-676 (1995); and
PCT International Publication No. 95/14098 (describing use of such
promoters in plants).
[0154] In embodiments, the promoter may be a WRKY76-specific
promoter cloned according to methods described herein.
[0155] The promoter may include, or be modified to include, one or
more enhancer elements to thereby provide for higher levels of
transcription. Examples of suitable enhancer elements for use in
plants described herein include, for example, the PC1SV enhancer
element, the CaMV 35S enhancer element and the FMV enhancer
element. See Maiti et al., Transgenic Res. 6:143-156 (1997); PCT
International Publication No. WO 96/23898; and U.S. Pat. Nos.
5,850,019; 5,106,739; and 5,164,316.
[0156] Expression Constructs
[0157] Embodiments include recombinant constructs comprising one or
more of the polynucleotide sequences described herein. The
constructs typically comprise a vector, such as a plasmid, a
cosmid, a phage, a virus, a bacterial artificial chromosome (BAC),
a yeast artificial chromosome (YAC), or the like, into which a
polynucleotide sequence as described herein has been inserted, in a
forward or reverse orientation. In some embodiments, the constructs
may further comprise regulatory sequences, including, e.g., a
promoter that is operably linked to the sequence. Vectors and
promoters suitable for recombinant constructs of the present
disclosure may include those generally known to persons having
skill in the art and/or described herein.
[0158] Constructs suitable for use in embodiments may contain a
"signal sequence" or "leader sequence" to facilitate
co-translational or post-translational transport of the polypeptide
of interest to certain intracellular structures, such as the
chloroplast (or other plastid), endoplasmic reticulum, or Golgi
apparatus, or to be secreted. Such sequences include leader
sequences targeting transport and/or glycosylation by passage into
the endoplasmic reticulum, vacuoles, plastids including
chloroplasts, mitochondria, and the like. For example, the
constructs may be engineered to contain a signal peptide to
facilitate transfer of the peptide to the endoplasmic reticulum. A
signal sequence is known or suspected to result in co-translational
or post-translational peptide transport across the cell membrane.
In eukaryotes, this typically involves secretion into the Golgi
apparatus, with some resulting glycosylation. Leader sequence
refers to any sequence that, when translated, results in an amino
acid sequence sufficient to trigger co-translational transport of
the peptide chain to a sub-cellular organelle. Plant expression
cassettes may also contain an intron, such that mRNA processing of
the intron is required for expression
[0159] Suitable constructs may also contain 5' and 3' untranslated
regions. A 3' untranslated region is a polynucleotide located
downstream of a coding sequence. Polyadenylation signal sequences
and other sequences encoding regulatory signals capable of
affecting the addition of polyadenylic acid tracts to the 3' end of
the mRNA precursor or the 3' untranslated regions. A 5'
untranslated region is a polynucleotide located upstream of a
coding sequence.
[0160] Any of the sequences described herein may be incorporated
into a cassette or vector for expression in plants. Expression
vectors suitable for stable transformation of plant cells or for
the establishment of transgenic plants are known by persons skilled
in the art. See e.g., Weissbach and Weissbach, Methods for Plant
Molecular Biology, Academic Press, (1989); and Gelvin et al., Plant
Molecular Biology Manual, Kluwer Academic Publishers (1990).
Specific examples may include those derived from a Ti plasmid of
Agrobacterium tumefaciens and those described for use in
dicotyledonous plants in Herrera-Estrella et al., Nature 303:209
(1983) and Klee, Bio/Technol. 3:637-642 (1985). In embodiments,
non-Ti vectors may be used to transfer the DNA into
monocotyledonous plants and cells by using free DNA delivery
techniques. Such methods may involve, for example, the use of
liposomes, electroporation, microprojectile bombardment, silicon
carbide whispers, and viruses.
[0161] The termination region may be native to the transcriptional
initiation region, the sequence described herein, or may be derived
from another source. Suitable termination regions may be derived
from the Ti-plasmid of A. tumefaciens, such as the octopine
synthase and nopaline synthase termination regions, or the
termination region of a plant gene, such as soybean storage
protein. See Guerineau et al., Mol. Gen. Genet. 262:141-144 (1991);
Proudfoot, Cell 64:671-674 (1991); Sanfacon et al., Genes Dev.
5:141-149 (1991); Mogen et al., Plant Cell 2:1261-1272 (1990);
Munroe et al., Gene 91:151-158 (1990); Ballas et al., Nucleic Acids
Res. 17:7891-7903 (1989); and Joshi et al., Nucleic Acids Res.
15:9627-9639 (1987). These vectors are plant integrating vectors in
that upon transformation, the vectors integrate a portion of vector
DNA into the genome of the host plant. An example of a vector
useful in embodiments is plasmid pBI121.
[0162] Where appropriate, the vector and WRKY76 transcription
factor sequences disclosed herein may be optimized for increased
expression in the transformed host cell. That is, the sequences may
be synthesized using host cell-preferred codons for improved
expression, or may be synthesized using codons at a host-preferred
codon usage frequency. Generally, the GC content of the
polynucleotide will be increased. See e.g., Campbell et al., Plant
Physiol. 92:1-11 (1990). Methods for synthesizing host-preferred
polynucleotides are known by persons skilled in the art. See e.g.,
Murray et al., Nucleic Acids Res. 17:477-498 (1989); U.S. Patent
Application Publications Nos. 2004/0005600 and 2001/0003849; and
U.S. Pat. Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, the
entire disclosures of which are incorporated by reference
herein.
[0163] For example, polynucleotides of interest can be targeted to
the chloroplast for expression. In this manner, where the
polynucleotide of interest is not directly inserted into the
chloroplast, an expression cassette according to some embodiments
may additionally contain a polynucleotide encoding a transcription
factor polypeptide to direct the nucleotide of interest to the
chloroplasts. Such transit peptides are known by persons skilled in
the art. See e.g., Von Heijne et al., Plant Mol. Biol. Rep.
9:104-126 (1991); Clark et al., J. Biol. Chem. 264:17544-17550
(1989); Della-Cioppa et al., Plant Phsyiol. 84:965-968 (1987);
Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421 (1993);
and Shah et al., Science 233:478-481 (1986). The polynucleotides of
interest to be targeted to the chloroplast may further be optimized
for expression in the chloroplast to account for differences in
codon usage between the plant nucleus and this organelle. In this
manner, the polynucleotides of interest may be synthesized using
chloroplast-preferred codons. See U.S. Pat. No. 5,380,831, the
entire disclosure of which is incorporated by reference herein.
[0164] In embodiments, one or more plant expression cassette (i.e.,
a WRKY76 transcription factor open reading frame operably linked to
a promoter) may be inserted into a plant transformation vector,
which allows for the transformation of DNA into a cell. Such
expression cassettes may be organized into more than one vector DNA
molecule.
[0165] Plant expression vectors suitable for use in embodiments may
comprise one or more DNA vector(s) for achieving plant
transformation. For example, it is common practice for persons
skilled in the art to utilize plant transformation vectors that
include one or more cloned plant coding sequences (genomic or cDNA)
under the transcriptional control of 5' and 3' regulatory sequences
as a dominant selectable marker. Such plant transformation vectors
typically also contain a promoter, a transcription initiation start
site, an RNA processing signal, a transcription termination site,
and/or a polyadenylation signal.
[0166] Binary vectors are plant transformation vectors that utilize
two non-contiguous DNA vectors to encode all requisite cis- and
trans-acting functions for transformation of plant cells. See
Hellens et al., Trends in Plant Sicence 5:446-451 (2000). Binary
vectors, as well as vectors with helper plasmids, are most often
used for Agrobacterium-mediated transformations, in which the size
and complexity of DNA segments needed to achieve efficient
transformation is large, and in which it is therefore advantageous
to separate functions among separate DNA molecules. Binary vectors
also typically contain a plasmid vector that contains the
cis-acting sequence required for T-DNA transfer (such as left
border and right border), a selectable marker that is engineered to
be capable of expression in a plant cell, and a polynucleotide of
interest (i.e., a polynucleotide engineered to be capable of
expression in a plant cell for which generation of transgenic
plants is desired). Sequences required for bacterial replication
may also be present on this plasmid vector. The cis-acting
sequences are arranged in a fashion to allow for efficient transfer
into plant cells and expression therein. For example, a selectable
marker sequence and a sequence of interest are typically located
between the left and right borders. Often a second plasmid vectors
the trans-acting factors that mediate T-DNA transfer from
Agrobacterium to the target (host) plant cells. This plasmid
typically contains virulence functions (Vir genes) that allow
infection of plant cells by Agrobacterium, and transfer of DNA by
cleavage at border sequences and vir-mediated DNA transfer, as
understood by persons skilled in the art. See e.g., Hellens et al.,
Trends in Plant Science 5:446-451 (2000). Several types of
Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.)
may be used for plant transformation. The second plasmid vector is
typically not necessary for introduction of polynucleotides into
plants by other methods, such as by microprojection,
microinjection, electroporation, etc.
[0167] In some embodiments, expression vectors include RNA
processing signals that can be positioned within, upstream or
downstream of the coding sequence. The expression vectors may
include additional regulatory sequences from the 3'-untranslated
region of plant genes. Initiation signals may also be used to aid
in efficient translation of coding sequences. These signals can
include, e.g., an ATG initiation codon and adjacent sequences. In
cases where a coding sequence, its initiation codon and upstream
sequences are inserted into the appropriate expression vector, no
additional translational control signals may be needed. However, in
cases where only coding sequences, or a portion thereof, are
inserted, exogenous transcriptional control signals including the
ATDG initiation codon can be separately provided. The initiation
codon is provided in the correct reading frame to facilitate
transcription. Exogenous transcription elements and initiation
codons can be of various origins, both natural and synthetic. The
efficiency of expression can be enhanced by including enhancers
appropriate for the cell system in use.
[0168] In embodiments where co-suppression of a gene is desired,
vectors in which RNA encoded by a transcription factor or
transcription factor homologue cDNA is over-expressed may be used
to obtain co-suppression of a corresponding endogenous gene. See
e.g., U.S. Pat. No. 5,231,020. Such co-suppression (also termed
"sense suppression") does not require that the entire transcription
factor cDNA be introduced into the plant cells, nor does it require
that the introduced sequence is identical to the endogenous
transcription factor gene of interest. However, as with antisense
suppression, the suppressive efficiency will be enhanced as the
specificity of hybridization is increased.
[0169] Embodiments include host (i.e., target) cells transduced
with vectors described herein, and the production of polypeptides
by recombinant techniques. Host cells are genetically engineered
(i.e., nucleic acids are introduced by transduction, transformation
or transfection) with the vectors, which may be, e.g., a cloning
vector or an expression vector comprising the relevant nucleic
acids described herein. The vector may optionally be, for example,
a plasmid, a viral particle, a phage, a naked nucleic acid,
etc.
[0170] Using polynucleotides disclosed herein, a protein may be
expressed in a recombinantly engineered cell, such as a plant cell.
Persons skilled in the art are knowledgeable about various
expression systems available for expression of a polynucleotide
encoding a protein according to embodiments described herein.
[0171] The expression of isolated polynucleotides encoding a
protein according to embodiments described herein will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter, followed by incorporation thereof into an expression
vector. Typical expression vectors contain transcription and
translation terminators, initiation sequences, and promoters useful
for regulation of the expression of the DNA encoding a protein
described herein. To obtain a high level of expression of a cloned
gene, it is typically desirable to construct expression vectors
that contain, at minimum, a strong promoter to direct
transcription, a ribosome binding site for translational
initiation, and a transcription/translation termination site.
Persons having ordinary skill in the art would recognize that
modifications could be made to a protein of the present disclosure
without diminishing its biological activity.
[0172] Embodiments and aspects of the invention include an
expression cassette. The expression cassette that comprises at
least: (1) a constitutive, inducible, or tissue-specific promoter;
and (2) a recombinant polynucleotide having a polynucleotide
sequence, or a complementary polynucleotide sequence thereof,
selected from the group consisting of a polynucleotide sequence
encoding: (a) a polypeptide sequence having a transcription factor
sequence as described herein; (b) a polynucleotide sequence
selected from the transcription factor polynucleotides described
herein; or sequence variants (e.g., allelic or splice variants) of
the polynucleotide sequences referenced in (a) or (b) above, where
the sequence variant encodes a polypeptide that regulates
transcription.
[0173] Expression Hosts
[0174] The invention also relates to host cells comprising
polynucleotides as described above, nucleic acid constructs,
vectors or expression cassettes as described above. For example,
the polynucleotide comprises or consists of SEQ ID NO:1 or a
variant thereof, or SEQ ID NO:9 or a variant thereof. In one
embodiment, the variant comprises or consists of SEQ ID NO:11.
[0175] In some embodiments, host cells are transduced with vectors
as described herein. Host cells are genetically engineered (i.e.,
nucleic acids are introduced, transformed or transfected) with the
vectors described herein, which may be, e.g., a cloning vector or
an expression vector comprising the relevant nucleic acids
disclosed herein. The vector is optionally a plasmid, a viral
particle, a phage, a nucleic acid, etc. The engineered host cells
can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying the relevant gene. The culture conditions, such as
temperature, pH and the like, may be those previously used with the
host cell selected for expression, and will be apparent to those
skilled in the art.
[0176] The host cell may be a eukaryotic cell, such as a yeast cell
or a plant cell, or the host cell may be a prokaryotic cell, such
as a bacterial cell, for example Agrobacterium. In some
embodiments, plant protoplasts may be suitable for use. In
preferred embodiments, the host cell is a plant cell. A kit
comprising such a host cell is also within the scope of the
invention.
[0177] For example, the DNA fragments may be introduced into plant
tissues, cultured plant cells or plant protoplasts by standard
methods, including, e.g., electroporation, infection by viral
vectors such as cauliflower mosaic virus (CaMV), high velocity
ballistic penetration by small particles with the nucleic acid
either within the matrix of small beads or particles, or on the
surface, use of pollen as a vector, or using Agrobacterium
tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA
fragments are cloned. See Fromm et al., Proc. Natl. Acad. Sci.
82:8524-5828 (1985); Hohn et al., Molecular Biology of Plant
Tumors, Academic Press, New York, N.Y. pp. 549-560 (1982); U.S.
Pat. No. 4,407,956; Klein et al., Nature 327:70-73 (1987); PCT
International Publication No. WO 85/01856. The T-DNA plasmid is
transmitted to plant cells upon infection by Agrobacterium
tumefaciens, and a portion is stably integrated into the plant
genome. See Horsch et al., Science 233:496-498 (1984); and Fraley
et al., Proc. Natl. Acad. Sci. 80:4803-4807 (1983).
[0178] The host cell may include a nucleic acid as described above.
In some embodiments the host cell may include a nucleic acid that
encodes a polypeptide according to embodiments described above,
such that the cell expresses a polypeptide of interest as described
herein. In some embodiments, the cell may include vector sequences
or the like. As will be understood by persons skilled in the art,
cells and transgenic plants that include any polypeptide or
polynucleotide sequence described herein (e.g., produced by
transduction of a vector described herein) represent embodiments
within the scope of the present disclosure.
[0179] For long-term, high-yield production of recombinant
proteins, stable expression may be used. Host cells transformed
with a nucleotide sequence encoding a polypeptide as disclosed
herein are optionally cultured under conditions suitable for the
expression and recovery of the encoded protein from cell culture.
The protein or fragment thereof produced by a recombinant cell may
be secreted, membrane-bound, or contained intracellularly,
depending on the sequence and/or the vector used. As will be
understood by persons skilled in the art, expression vectors
according to embodiments containing polynucleotides that encode
mature proteins can be designed with signal sequences that direct
secretion of the mature polypeptides through a prokaryotic or
eukaryotic cell membrane.
[0180] Some embodiments of the invention relate to recombinant
expression of a WRKY76 transcription factor protein in a stable
cell line. Methods for generating a stable cell line following
transformation of a heterologous construct into a host cell are
known in the art. The transformed cells, tissues, and plants are
therefore understood to encompass not only the end product of a
transformation process, but also transgenic progeny or propagated
forms thereof.
[0181] Transgenic Plants and Production Thereof
[0182] Embodiments also relate to transgenic plants, methods of
producing the transgenic plants, and methods for modifying plant
traits or conferring desirable traits upon host plants to produce
transgenic plants having improved stress tolerance and yield in
standard growing conditions. Also within the scope of the invention
are plants obtained or obtainable by the methods described
herein.
[0183] Polynucleotides disclosed herein are favorably employed to
produce transgenic plants with various traits or characteristics
that have been modified in a desirable manner, e.g., to improve the
seed characteristics of the plant. For example, altering the
expression levels or patterns of one or more of the transcription
factors (or transcription factor homologues) disclosed herein, as
compared with the levels of the same protein found in a control
wild-type plant, can be used to modify a plant's traits.
Illustrative examples of trait modification and improved
characteristics resulting from altering expression levels of the
disclosed WRKY76 transcription factor sequences are explained in
more detail in the various Examples below.
[0184] In some aspects of the invention, plants expressing a
heterologous WRKY76 transcription factor, including plants that
express a WRKY76 transcription factor at elevated levels, are
provided. Still other aspects of the invention relate to the
generation of plants with conditional or inducible expression of a
WKKY transcription factor protein as disclosed herein.
[0185] Thus, in one further aspect, the invention relates to a
transgenic plant comprising and expressing a nucleic acid
construct, vector or expression cassette comprising a nucleic acid
that encodes a WRKY76 transcription factor. For example, said
nucleic acid comprises or consists of SEQ ID NO:1 or a variant
thereof, for example SEQ ID NO:11, or SEQ ID NO:9, a functional
variant or part thereof as defined above. In one embodiment, said
polynucleotide has at least 80% sequence identity with the
full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO:11. In another embodiment, said
polynucleotide has at least 80% sequence identity with the
full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO:11, wherein the polynucleotide
contains at least one of: [0186] (a) adenine as the nucleotide
corresponding to position 817 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO:11; [0187] (b) cytosine as the nucleotide corresponding to
position 808 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO:11; [0188] (c) cytosine
as the nucleotide corresponding to position 33 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO:11; [0189] (d) cytosine as the nucleotide
corresponding to position 259 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO:11; and [0190] (e) guanine as the nucleotide corresponding to
position 315 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO:11.
[0191] A plant according to the various aspects of the invention,
including the transgenic plants, methods and uses described herein
may be a monocot or a dicot plant. A dicot plant may be selected
from the families including, but not limited to Asteraceae,
Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae,
Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae,
Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For
example, the plant may be selected from lettuce, sunflower,
Arabidopsis, broccoli, spinach, water melon, squash, cabbage,
tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose,
strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil,
peanut, chickpea, apricots, pears, peach, grape vine, bell pepper,
chilli or citrus species. A monocot plant may, for example, be
selected from the families Arecaceae, Amaryllidaceae or Poaceae.
For example, the plant may be a cereal crop, such as maize, wheat,
rice, barley, oat, sorghum, rye, millet, buckwheat, or a grass crop
such as Lolium species or Festuca species, or a crop such as sugar
cane, onion, leek, yam or banana. Also included are biofuel and
bioenergy crops such as rape/canola, sugar cane, sweet sorghum,
Panicum virgatum (switchgrass), linseed, lupin and willow, poplar,
poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine.
Also included are crops for silage (maize), grazing or fodder
(grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax),
building materials (e.g. pine, oak), pulping (e.g. poplar), feeder
stocks for the chemical industry (e.g. high erucic acid oil seed
rape, linseed) and for amenity purposes (e.g. turf grasses for golf
courses), ornamentals for public and private gardens (e.g.
snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and
cut flowers for the home (African violets, Begonias,
chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber
plant). Preferably, the plant is a crop plant. By crop plant is
meant any plant which is grown on a commercial scale for human or
animal consumption or use. In a preferred embodiment, the plant is
a cereal. Most preferred plants are maize, rice, wheat, oilseed
rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato,
tobacco, grape, barley, pea, bean, field bean, lettuce, cotton,
sugar cane, sugar beet, broccoli or other vegetable brassicas or
poplar.
[0192] The term "plant" as used herein encompasses whole plants,
ancestors and progeny of the plants and plant parts, including
seeds, fruit, shoots, stems, leaves, roots (including tubers),
flowers, tissues and organs, wherein each of the aforementioned
comprise the nucleic acid construct as described herein. The term
"plant" also encompasses plant cells, suspension cultures, callus
tissue, embryos, meristematic regions, gametophytes, sporophytes,
pollen and microspores, again wherein each of the aforementioned
comprises the nucleic acid construct as described herein.
[0193] The aspects of the invention also extend to products
derived, preferably directly derived, from a harvestable part of
such a plant, such as dry pellets or powders, oil, fat and fatty
acids, starch or proteins. The invention also relates to food
products and food supplements comprising the plant of the invention
or parts thereof.
[0194] The transgenic plants show increased tolerance to stress
conditions, for example abiotic and biotic stress compared to a
control plant, for example a wild type plant. In particular, the
plants show increased stress response to abiotic stress selected
from drought or irrigation.
[0195] Plants expressing a heterologous WRKY76 transcription factor
protein may be further modified at more than one WRKY76
transcription factor locus or at a locus other than a WRKY76
transcription factor locus to confer increased stress tolerance or
another trait of interest.
[0196] The invention also relates to method for producing
transgenic plants comprising introducing and expressing in a plant
a polynucleotide, vector or expression cassette as described above
or a polynucleotide encoding a WRKY76 polypeptide as described
above. Such methods comprise generating from the plant cell a
transgenic plant that expresses the polynucleotide. In one
embodiment, the method comprises introducing and expressing a
nucleic acid that comprises or consists of SEQ ID NO:1, a
functional variant or part thereof as defined above. In one
embodiment, said polynucleotide has at least 80% sequence identity
with the full-length nucleotide sequence of SEQ ID NO: 1. In
another embodiment, said polynucleotide has at least 80% sequence
identity with the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO:11 or SEQ ID NO:9,
wherein the polynucleotide contains at least one of: [0197] (a)
adenine as the nucleotide corresponding to position 817 of the
full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO:11; [0198] (b) cytosine as the
nucleotide corresponding to position 808 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO:11; [0199] (c) cytosine as the nucleotide
corresponding to position 33 of the full-length nucleotide sequence
of SEQ ID NO: 1 or a variant thereof, for example SEQ ID NO:11;
[0200] (d) cytosine as the nucleotide corresponding to position 259
of the full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO:11; and [0201] (e) guanine as the
nucleotide corresponding to position 315 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO:11.
[0202] To prepare a plant expressing a heterologous WRKY76
transcription factor according to embodiments, the introduction of
a WKKY polynucleotide disclosed herein may be accomplished by
techniques known in the art, including, but not limited to,
electroporation or chemical transformation. See e.g., Ausubel,
Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,
Indianapolis, Ind. (1994). Markers conferring resistance to toxic
substances may also be used to identify transformed cells (having
taken up and expressed the test polynucleotide sequence) from
non-transformed cells (those not containing or not expressing the
test polynucleotide sequence). The stable transformation of
"transformed" or "transgenic" plants as used herein refers to the
introduction of a polynucleotide construct into a plant, such that
it integrates into the genome of the plant and is capable of being
inherited by progeny thereof.
[0203] In general, plant transformation methods involve
transferring heterologous DNA into target plant cells, followed by
applying a maximum threshold level of appropriate selection to
recover the transformed plant cells from an untransformed cell
mass. Subsequently, the transformed cells are differentiated into
shoots after being placed on a regeneration medium supplemented
with a maximum threshold level of selecting agent (e.g.,
temperature, herbicide, etc.). The shoots are then transferred to a
selective rooting medium for recovering the rooted shoot or
plantlet. The transgenic plantlet is then grown into a mature plant
that produces fertile seeds. A general description of techniques
and methods for generating transgenic plants may be found in Ayres
et al., CRC Crit. Rev. Plant Sci. 13:219-239 (1994), and Bommineni
et al., Maydica 42:107-120 (1997).
[0204] In general, since the transformed material contains many
cells, both transformed and non-transformed cells are present in
any piece of subjected target callus, tissue, or group of cells.
The ability to kill non-transformed cells and allow transformed
cells to proliferate results in transformed plant cultures. Often,
the ability to remove non-transformed cells is a limitation to
rapid recovery of transformed plant cells and successful generation
of transgenic plants. Therefore, molecular and biochemical methods
may be used for confirming the presence of the integrated
nucleotide(s) of interest in the genome of the transgenic plant.
For example, selectable markers, such as enzymes resulting in a
change of color or luminescent molecules (e.g., GUS and
luciferase), antibiotic-resistant genes (e.g., gentamicin and
kanamycin-resistance genes) and chemical-resistant genes (e.g.,
herbicide-resistance genes) may be used to confirm the integration
of the nucleotide(s) of interest in the genome of the transgenic
plant. Alternatively, particularly in considering the safety of the
transgenic plants, the transformed plants can be selected under
environmental stresses avoiding the incorporation of any selectable
marker genes.
[0205] Transformation and regeneration of both monocotyledonous and
dicotyledonous plant cells has become routine, and the selection of
the most appropriate transformation technique can be readily
determined by the person skilled in the art. The choice of method
will typically vary based on the type of plant to be transformed,
as persons skilled in the art will recognize the suitability of
particular methods for given plant types. Suitable methods for use
in embodiments may include, but are not limited to: electroporation
of plant protoplasts; liposome-mediated transformation;
polyethylene glycol (PEG) mediated transformation; transformation
using viruses; micro-injection of plant cells; micro-projectile
bombardment of plant cells; vacuum infiltration; and Agrobacterium
tumefaciens mediated transformation. Successful examples of
modifications of plant characteristics by transformation with
cloned sequences, which serve to illustrate current knowledge in
the art and are herein incorporated by reference, include: U.S.
Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945;
5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269;
5,736,369; and 5,610,042.
[0206] The generation of transgenic plants according to embodiments
may be performed by methods known to persons skilled in the art,
including: introduction of heterologous DNA by Agrobacterium into
plant cells (Agrobacterium-mediated transformation); bombardment of
plant cells with heterologous foreign DNA adhered to particles; and
various other non-particle direct-mediated methods, such as
microinjection, electroporation, application of Ti plasmid, Ri
plasmid, or plant virus vector, and direct DNA transformation.
[0207] Generally, there are three types of conventional
Agrobacterium-mediated transformation methods. The first method
involves co-cultivation of Agrobacterium with cultured isolated
protoplasts. This method requires an established culture system
that allows culturing protoplasts and plant regeneration from
cultured protoplasts. The second method involves transformation of
cells or tissues with Agrobacterium. This method requires that the
plant cells or tissues can be transformed by Agrobacterium, and
that the transformed cells or tissues can be induced to regenerate
into whole plants. The third method involves the transformation of
seeds, apices or meristems with Agrobacterium. This method requires
micropropagation.
[0208] The efficiency of Agrobacterium-mediated transformation
methods may be enhanced by, e.g., including in the Agrobacterium
culture a natural wound response molecule, such as acetosyringone
(AS), which has been shown to enhance transformation efficiency
with Agrobacterium tumefaciens. See Shahla et al., Plant Molec.
Biol. 8:291-298 (1987). Alternatively, transformation efficiency
may be enhanced by wounding the target tissue to be transformed by,
e.g., punching, maceration, bombardment with microprojectiles, etc.
See e.g., Bidney et al., Plant Molec. Biol. 18:301-313 (1992).
[0209] In some embodiments, plant cells may be transfected with
vectors via particle bombardment (e.g., with a gene gun). Particle
mediated gene transfer methods are known in the art, are
commercially available, and include, e.g., the gas driven gene
delivery instrument described in U.S. Pat. No. 5,584,807, the
contents of which are incorporated by reference herein. This method
involves coating the polynucleotide sequence of interest onto heavy
metal particles, and accelerating the coated particles under the
pressure of compressed gas for delivery to the target tissue.
[0210] In some embodiments, specific initiation signals may be used
to achieve more efficient translation of sequences encoding a
polypeptide described herein, such as, e.g., the ATG initiation
codon and adjacent sequences. In cases where sequences encoding the
polypeptide of interest, its initiation codon, and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However in cases where only the coding sequence or a
portion thereof is inserted, heterologous translational control
signals that include the ATG initiation codon may be provided.
[0211] In addition to expression of nucleic acids disclosed herein
as gene replacement or plant phenotype modification nucleic acids,
disclosed nucleic acids may also be used for sense and anti-sense
suppression of expression, e.g., to down-regulate expression of the
disclosed nucleic acids, as a further mechanism for modulating
plant phenotypes. That is, nucleic acids described herein, or
subsequences or anti-sense sequences thereof, can be used to block
expression of naturally occurring homologous nucleic acids. A
variety of sense and anti-sense technologies is known in the art.
See e.g., Lichtenstein and Nellen, Antisense Technology: A
Practical Approach, IRL Press at Oxford University, Oxford, England
(1997). In general, sense or anti-sense sequences are introduced
into a cell, where they are optionally amplified, e.g., by
transcription. Such sequences include both simple oligonucleotide
sequences and catalytic sequences, such as ribozymes.
[0212] For example, the reduction or elimination of expression
(i.e., a "knock-out") of a transcription factor or transcription
factor homologue polypeptide in a transgenic plant, e.g., to modify
a plant trait, can be obtained by introducing an antisense
construct corresponding to the polypeptide of interest as a cDNA.
For antisense suppression, the transcription factor or homologue
cDNA is arranged in reverse orientation (with respect to the coding
sequence) relative to the promoter sequence in the expression
vector. The introduced sequence need not be the full length cDNA or
gene, and need not be identical to the cDNA or gene found in the
plant type to be transformed. Typically, the antisense sequence
need only be capable of hybridizing to the target gene or RNA or
interest. Thus, where the introduced sequence is of shorter length,
a higher degree of homology to the endogenous transcription factor
sequence will be needed for effective antisense suppression. While
antisense sequences of various lengths can be utilized, preferably,
the introduced antisense sequence in the vector will be at least 30
nucleotides in length, and improved antisense suppression will
typically be observed as the length of the antisense sequence
increases. Preferably, the length of the antisense sequence in the
vector will be greater than 100 nucleotides. Transcription of an
antisense construct as described herein results in the production
of RNA molecules that are the reverse complement of mRNA molecules
transcribed form the endogenous transcription factor gene in the
plant cell.
[0213] In some embodiments, suppression of endogenous transcription
factor gene expression can also be achieved using a ribozyme.
Ribozymes are RNA molecules that possess highly specific
endoribonuclease activity. The production and use of ribozymes are
disclosed in U.S. Pat. Nos. 4,987,071 and 5,543,508. Synthetic
ribozyme sequences including antisense RNAs can be used to confer
RNA cleaving activity on the antisense RNA, such that endogenous
mRNA molecules that hybridize to the antisense RNA are cleaved,
which in turn leads to an enhanced antisense inhibition of
endogenous gene expression.
[0214] In some embodiments, vectors in which RNA encoded by a
transcription factor or transcription factor homologue cDNA is
over-expressed may be used to obtain co-suppression of a
corresponding endogenous gene, as described in U.S. Pat. No.
5,231,020. Such co-suppression (also termed "sense suppression")
does not require that the entire transcription factor cDNA to be
introduced into the plant cell, nor does it require that the
introduced sequence be exactly identical to the endogenous
transcription factor gene of interest. However, as with antisense
suppression, the suppressive efficiency will be enhanced as
specificity of hybridization is increased, e.g., as the introduced
sequence is lengthened, and/or as the sequence similarity between
the introduced sequence and the endogenous transcription factor
gene is increased.
[0215] Vectors expressing an untranslatable form of the
transcription factor mRNA, e.g., sequences comprising one or more
stop codon, or nonsense mutation, can also be used to suppress
expression of an endogenous transcription factor, thereby reducing
or eliminating its activity and modifying one or more traits.
Methods for producing such constructs are described in U.S. Pat.
No. 5,583,021. Preferably, such constructs are made by introducing
a premature stop codon into the transcription factor gene.
[0216] Another method for abolishing the expression of a gene is by
insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens.
After generating the insertion mutants, the mutants can be screened
to identify those containing the insertion in a transcription
factor or transcription factor homologue gene. Plants containing a
single transgene insertion even at the desired gene can be crossed
to generate homozygous plants for the mutation. See Koncz et al.,
Methods in Arabidopsis Research, World Scientific (1992).
[0217] Alternatively, a plant phenotype can be altered by
eliminating an endogenous gene, such as a transcription factor or
transcription factor homologue, e.g., by homologous recombination.
See Kempin et al., Nature, 389:820 (1997).
[0218] Polynucleotides and polypeptides disclosed herein can also
be expressed in a plant in the absence of an expression cassette by
manipulating the activity or expression level of the endogenous
gene by other means, e.g., by ectopically expressing a gene by
T-DNA activation tagging. See Ichikawa et al., Nature 390:698-701
(1997); and Kakimoto et al., Science 274:982-985 (1996). This
method entails transforming a plant with a gene tag containing
multiple transcriptional enhancers and, once the tag has inserted
into the genome, expression of a flanking gene coding sequence
becomes deregulated. As another example, the transcriptional
machinery in a plant can be modified so as to increase
transcription levels of a polynucleotide disclosed herein. See
e.g., PCT International Publications Nos. WO 96/06166 and WO
98/53057 (describing modifications of DNA binding specificity of
zinc finger proteins by changing particular amino acids in the DNA
binding motif).
[0219] Following transformation, the plants are preferably selected
using a dominant selectable marker incorporated into the
transformation vector. After transformed plants are selected and
grown to maturity, those plants showing a modified trait are
identified. The modified train can be any of the traits described
herein. Additionally, to confirm that the modified trait is due to
changes in expression levels or activity of the polypeptide or
polynucleotide disclosed herein, the mRNA expression may be
analyzed using Northern blots, RT-PCR or microarrays, or protein
expression using immunoblots, Western blots, or gel shift
assays.
[0220] In some embodiments, the plants may be homozygous for the
WRKY76 polynucleotide disclosed herein, i.e., a transgenic plant
that contains two added sequences, one sequence at the same locus
on each chromosome of a chromosome pair. A homozygous transgenic
plant according to these embodiments can be obtained by sexually
mating (selfing) an independent segregating transgenic plant that
contains the added sequences disclosed herein, germinating some of
the seed produced and analyzing the resulting plants produced for
enhanced enzyme activity and/or increased plant yield relative to a
control (native, non-transgenic) or an independent segregant
transgenic plant. Persons skilled in the art will understand that
two different transgenic plants may be mated to produce offspring
that contain two independently segregating added heterologous
polynucleotides.
[0221] Cells that have been transformed may be grown into plants in
conventional ways. See e.g., McCormick et al., Plant Cell Rep.
5:81-84 (1986). The plants may be grown, and either pollinated with
the same transformed strain or different strains, the resulting
hybrid having constitutive expression of the desired phenotypic
characteristic identified. Two or more generations may be grown to
ensure that expression of the desired phenotypic characteristic is
stably maintained and inherited, and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved. In this manner, the present disclosure provides for
transformed seeds (also referred to as transgenic seeds) having a
polynucleotide as disclosed herein (e.g., an expression cassette as
disclosed herein) stably incorporated into their genome.
[0222] Methods for Modulating a Plant Phenotype
[0223] In another aspect, the invention relate to a method for
modulating a plant phenotype comprising introducing and expressing
in a plant a polynucleotide, vector or expression cassette as
described above or a polynucleotide encoding a WRKY76 polypeptide
as described above. Such methods comprise generating from the plant
cell a transgenic plant that expresses the polynucleotide. In one
embodiment, the method comprises introducing and expressing a
nucleic acid that comprises or consists of SEQ ID NO:1 or a variant
thereof, for example SEQ ID NO:11 or SEQ ID NO:9, a functional
variant or part thereof as defined above. In one embodiment, said
polynucleotide has at least 80% sequence identity with the
full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO:11. In another embodiment, said
polynucleotide has at least 80% sequence identity with the
full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO:11, wherein the polynucleotide
contains at least one of: [0224] (a) adenine as the nucleotide
corresponding to position 817 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO:11; [0225] (b) cytosine as the nucleotide corresponding to
position 808 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO:11; [0226] (c) cytosine
as the nucleotide corresponding to position 33 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO:11; [0227] (d) cytosine as the nucleotide
corresponding to position 259 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO:11; and [0228] (e) guanine as the nucleotide corresponding to
position 315 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO:11.
[0229] The inventors have surprisingly demonstrated that expressing
HaKRKY76 increases yield under standard growth conditions and also
increases stress tolerance under mild and severe conditions. Thus,
in one embodiment, said phenotype is increased yield and said
method is directed to increasing yield of a plant compared to a
control plant.
[0230] The term "yield" includes one or more of the following
non-limitative list of features: early flowering time, biomass
(vegetative biomass (root and/or shoot biomass) or seed/grain
biomass), seed/grain yield, seed/grain viability and germination
efficiency, seed/grain size, starch content of grain, early vigour,
greenness index, increased growth rate, delayed senescence of green
tissue. The term "yield" in general means a measurable produce of
economic value, typically related to a specified crop, to an area,
and to a period of time. Individual plant parts directly contribute
to yield based on their number, size and/or weight. The actual
yield is the yield per square meter for a crop and year, which is
determined by dividing total production (includes both harvested
and appraised production) by planted square meters.
[0231] Thus, according to the invention, yield comprises one or
more of and can be measured by assessing one or more of: increased
seed yield per plant, increased seed filling rate, increased number
of filled seeds, increased harvest index, increased
viability/germination efficiency, increased number or size of
seeds/capsules/pods/grain, increased growth or increased branching,
for example inflorescences with more branches, increased biomass or
grain fill. Preferably, increased yield comprises an increased
number of grain/seed/capsules/pods, increased biomass, increased
growth, increased number of floral organs and/or floral increased
branching. Yield is increased relative to a control plant. For
example, the yield is increased by 2%, 3%, 4%, 5%-10%, 10%-50% or
more compared to a control plant, for example by at least 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45% or 50%.
[0232] In another embodiment, said phenotype is stress tolerance
and said method relates to increasing stress tolerance of a plant.
The term "stress" or "stress condition" as used herein includes
abiotic and biotic stress. Said stress/stress tolerance is
preferably selected from one or any combination of the following:
freezing, low temperature, chilling, drought, high salinity,
waterlogging. In one preferred embodiment, the stress is
drought.
[0233] Species such as winter cereals are adapted to cold or
moderate-cold weather and can tolerate temperatures ranging from
0.degree. C. to 15.degree. C., as well as freezing temperatures,
rather well if they have previously been acclimated to reduced
temperatures. By contrast, tropical and subtropical species,
including important crops, such as maize, rice or tomato, are
sensitive to low temperatures and appear to lack efficient
acclimation mechanisms.
[0234] Furthermore, in Arabidopsis research, stress is often
assessed under severe conditions that are lethal to wild type
plants. For example, drought tolerance is assessed predominantly
under quite severe conditions in which plant survival is scored
after a prolonged period of soil drying. However, in temperate
climates, limited water availability rarely causes plant death, but
restricts biomass and seed yield. Moderate water stress, that is
suboptimal availability of water for growth can occur during
intermittent intervals of days or weeks between irrigation events
and may limit leaf growth, light interception, photosynthesis and
hence yield potential. Leaf growth inhibition by water stress is
particularly undesirable during early establishment.
[0235] In Skirycz et al., 2011, different transgenic Arabidopsis
events with enhanced tolerance to lethal drought were analyzed in a
mild stress assay. The authors screened the literature in order to
identify Arabidopsis genes that in gain- or loss-of-function
situations confer drought stress tolerance without penalties in
growth, and then selected 25 to perform the assay. In this assay,
two lines showed larger plants while the rest were smaller, either
in control or under drought conditions. However, growth reduction
under mild stress was similar for all of the genotypes tested. The
authors therefore concluded that enhanced survival under severe
drought is not a good indicator for improved growth performance
under mild/moderate stress conditions which can often be found in
temperate climates. Superior survival under severe drought is often
associated with constitutive activation of water-saving mechanisms,
such as stomatal closure, that can lead to growth penalty.
Therefore, genes that are useful in conferring tolerance to severe
stress conditions in transgenic plants and increase survival rates
are in most cases detrimental to plant yield when the transgenic
plant expressing such transgene(s) is exposed to mild stress
conditions.
[0236] The terms moderate or mild stress/stress conditions are used
interchangeably and refer to non-severe stress non-lethal stress.
Moderate stress, unlike severe stress, does not lead to plant
death. Under moderate, that is non-lethal, stress conditions, wild
type plants are able to survive, but show a decrease in growth and
seed production and prolonged moderate stress can also result in
developmental arrest. The decrease can be at least 5%-50% or more.
The effects of severe stress are usually measured as % tage of
surviving plants, whereas the effects of moderate stress can be
measured by assessing yield, growth or other parameters other than
plant survival. Tolerance to severe stress is measured as a
percentage of survival, whereas moderate stress does not affect
survival, but growth rates.
[0237] Accordingly, the term moderate stress as used herein results
in a measurable decrease of growth rates in wild type plants.
Assays that mimic moderate stress conditions for Arabidopsis
thaliana plants are described herein and in Skirycz et al, 2011.
The decrease may be at least 5%-50% or more, for example 5%-10%,
1-25%, 20-30%, 30-40%, 40-50%.
[0238] The precise conditions that define moderate stress vary from
plant to plant and also between climate zones, but ultimately,
these moderate conditions do not cause the plant to die. With
regard to high salinity for example, most plants can tolerate and
survive about 4 to 8 dS/m. Specifically, in rice, soil salinity
beyond ECe.about.4 dS/m is considered moderate salinity while more
than 8 dS/m becomes high. Similarly, pH 8.8-9.2 is considered as
non-stress while 9.3-9.7 as moderate stress and equal or greater
than 9.8 as higher stress.
[0239] Drought stress can be measured through leaf water
potentials. Generally speaking, moderate drought stress is defined
by a water potential of between -1 and -2 Mpa. This has for example
been applied in experiments relating to barley and Phaseolus
vulgaris L. (Wingler et al, 1999 and Torres-Franklin et al
2007).
[0240] Waterlogging/irrigation: stress. Waterlogging is flooding of
the root system whereas submergence is related to immersion of the
whole plant (Bailey-Serres et al, Trends in Plant Sciences, 2012,
Vo. 17, No. 3, 129-138).
[0241] Moderate temperatures vary from plant to plant and specially
between species. Normal temperature growth conditions for
Arabidopsis are defined at 22-24.degree. C. For example, at
28.degree. C., Arabidopsis plants grow and survive, but show severe
penalties because of "high" temperature stress associated with
prolonged exposure to this temperature. However, the same
temperature of 28.degree. C. is optimal for sunflower, a species
for which 22.degree. C. or 38.degree. C. causes mild, but not
lethal stress. In other words, for each species and genotype, an
optimal temperature range can be defined as well as a temperature
range that induces mild stress or severe stress which leads to
lethality.
[0242] Also, depending on the soil conditions and/or geographic
region in which the plant is grown, "mild stress conditions" can be
constant/permanent. For example, the yield of the same soybean (or
maize) genotype exhibits differences every year when comparing
different regions presenting varied rainfall regimes, even when no
drought season occurred during this time.
[0243] Moderate stress conditions are common even in temperate
climates and affect yield. A skilled person would be able to
determine temperatures that can lead to mild stress for any given
species based on common general knowledge in the technical field
and/or routine methods.
[0244] Specifically, according of the aspects of the invention,
HaWRKY76 can be expressed in another plant that is not sunflower to
elicit the beneficial effects described herein.
[0245] In other aspects, the invention relates to plants or parts
thereof obtained or obtainable by the methods described herein as
well as products derived therefrom.
[0246] In yet another aspect, the invention relates to the use of a
nucleic acid described above in increasing tolerance to abiotic or
biotic stress. In one embodiment, said nucleic acid comprises or
consists of SEQ ID NO:1 or a variant thereof, for example SEQ ID
NO: 11 or SEQ ID NO:9, a functional variant or part thereof as
defined above. In one embodiment, said polynucleotide has at least
80% sequence identity with the full-length nucleotide sequence of
SEQ ID NO: 1 or SEQ ID NO:9. In another embodiment, said
polynucleotide has at least 80% sequence identity with the
full-length nucleotide sequence of SEQ ID NO: 1 or a variant
thereof, for example SEQ ID NO:11, wherein the polynucleotide
contains at least one of: [0247] (a) adenine as the nucleotide
corresponding to position 817 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO:11; [0248] (b) cytosine as the nucleotide corresponding to
position 808 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO:11; [0249] (c) cytosine
as the nucleotide corresponding to position 33 of the full-length
nucleotide sequence of SEQ ID NO: 1 or a variant thereof, for
example SEQ ID NO:11; [0250] (d) cytosine as the nucleotide
corresponding to position 259 of the full-length nucleotide
sequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID
NO:11; and [0251] (e) guanine as the nucleotide corresponding to
position 315 of the full-length nucleotide sequence of SEQ ID NO: 1
or a variant thereof, for example SEQ ID NO:11.
[0252] In one embodiment, said stress is abiotic stress and for
example selected from drought or irrigation. In one embodiment,
said stress is moderate stress. In another embodiment, said stress
is severe stress.
[0253] In another aspect, the invention relates to an isolated
HaWRKY76 promoter nucleic acid sequence. This can be selected from
SEQ ID NO: 10 or a sequence with at least 80%, 85%, 90%, or 95%
sequence identity with SEQ ID NO: 10. In one embodiment, the
sequence does not comprise the 5' UTR. In another aspect, the
invention relates to a vector comprising SEQ ID NO: 10 or a
sequence with at least 80%, 85%, 90%, or 95% sequence identity with
SEQ ID NO: 10. Also within the scope of the invention is plant
expressing a construct comprising a nucleic acid molecule
comprising SEQ ID NO: 10 or a sequence with at least 80%, 85%, 90%,
or 95% sequence identity with SEQ ID NO: 10 operably linked to
another nucleic acid sequence, a method for making such plants and
uses thereof in controlling stress responses.
[0254] Specific embodiments are further described by the following
Examples.
[0255] While the foregoing disclosure provides a general
description of the subject matter encompassed within the scope of
the present invention, including methods, as well as the best mode
thereof, of making and using this invention, the following examples
are provided to further enable those skilled in the art to practice
this invention and to provide a complete written description
thereof. However, those skilled in the art will appreciate that the
specifics of these examples should not be read as limiting on the
invention, the scope of which should be apprehended from the claims
and equivalents thereof appended to this disclosure. Various
further aspects and embodiments of the present invention will be
apparent to those skilled in the art in view of the present
disclosure. All documents mentioned in this specification are
incorporated herein by reference in their entirety.
[0256] "and/or" where used herein is to be taken as specific
disclosure of each of the two specified features or components with
or without the other. For example "A and/or B" is to be taken as
specific disclosure of each of (i) A, (ii) B and (iii) A and B,
just as if each is set out individually herein. Unless context
dictates otherwise, the descriptions and definitions of the
features set out above are not limited to any particular aspect or
embodiment of the invention and apply equally to all aspects and
embodiments which are described. The application claims benefit
from U.S. 61/986,730. HaWRKY76 is termed HaWKKY2 therein.
Accordingly, the terms HaWRKY76 and HaWKKY2 are synonymous and are
used interchangeably.
EXAMPLES
[0257] In reference to the following Examples, it will be
recognized by persons skilled in the art that a transcription
factor that is associated with a particular first trait may also be
associated with at least one other, unrelated and inherent second
trait which is not predicted by the first trait.
[0258] Genetic Construct
[0259] 35SCaMV:HaWRKY76 was employed in the various Examples
described below. The HaWRKY76 coding sequence was obtained from
Helianthus annuus CF31 genotype. It was amplified by RT-PCR with
specific oligonucleotides using as a template the total RNA of the
leaves. The amplification product was cloned directly in the pBI121
vector. In this way, the cDNA expression was controlled by the 35S
CaMV promoter. The construct 35S:HaWRKY76 was initially introduced
in the BL21 (DE3) E. coli strain and then transferred to
Agrobacterium tumefaciens strain LBA4404 by electroporation using
the GENE PULSER.TM. (Bio-Rad). The protein sequence as shown in SEQ
ID NO:12 was expressed in plants.
[0260] Plant Material and Growth Conditions
[0261] Arabidopsis thaliana ecotype Columbia (Col-0) was purchased
from Lehle Seeds (Tucson, Ariz.). Plants were grown directly on
soil in a growth chamber at 22-24.degree. C. under long-day
photoperiods (16 hours of illumination with a mixture of cool-white
and GroLux fluorescent lamps) at an intensity of approximately 180
.mu.E m.sup.-2s.sup.-1 in pots (8 cm diameter.times.7 cm
height).
[0262] Seeds of Helianthus annuus L. (sunflower CF31, from Advanta
Seeds) were grown in the soil pots in a culture chamber at
28.degree. C. for variable periods of time depending on the purpose
of the experiment, as further detailed in the figure legends.
[0263] Drought Assays
[0264] Depending on the particular Experiment, as detailed in the
figure legends, sunflower seedlings were placed on a filter paper
during 30 minutes, and R2 plants grown in soil pots were stressed
by stopping watering (dehydration) during 15 days. Every one, two,
or three days after this treatment, 1 cm diameter leaf disks were
frozen in liquid nitrogen for further RNA analysis.
[0265] As a control of viability of the plants, plants were
re-watered after the taking of the sample. Only the samples of the
survivors were analyzed.
[0266] 25-day-old Arabidopsis plants were subjected to drought
stress by stopping of watering (dehydration) during 16-18 days.
After this treatment, the plants were re-watered to saturation.
Survivor plants were counted two days after recovery at normal
growth conditions.
[0267] In the case of drought assays maintaining the same water
volume, soil pots were weighed every 2-3 days, and water was added
to those pots that needed watering in order to maintain the same
weight in each soil pot. The weight differences were recorded so as
to calculate the total water volume added to each pot.
[0268] Transformation of Arabidopsis thaliana
[0269] Transformed Agrobacterium tumefaciens strain LBA4404 was
used to obtain transgenic Arabidopsis plants. The method employed
for transforming Arabidopsis thaliana was the one using immersion
(the floral dip procedure), as describe by Clough and Bent, Plant
J. 16:735-743 (1998). Transformed plants were selected on the basis
of kanamycin resistance and positive PCR, which was carried out on
genomic DNA with specific oligonucleotides. Five positive
independent lines were used to select homozygous T3 and T4 in order
to analyze phenotypes and the expression levels of HaWRKY76. To
assess HaWRKY76 expression, real time RT-PCR was performed on
homozygous transformants, as described further below. Plants
transformed with pBI101.3 were used as negative controls.
[0270] RNA Isolation and Expression Analyses by Real Time
RT-PCR
[0271] RNA for real time RT-PCR was prepared with TRIZOL.RTM.
reagent (Invitrogen.TM.) according to the manufacturer's
instructions. RNA (2 .mu.g) was used for the RT-PCR reactions using
M-MLV reverse transcriptase (Promega). Quantitative PCRs were
carried out using a MJ-Chromo4 apparatus in a 20 .mu.l final volume
containing 1 .mu.l SyBr green (10.times.), 9 pmol of each primer, 2
mM MgCl.sub.2, 10 .mu.l of a 1/30 dilution of the RT-PCR reaction,
and 0.05 .mu.k Platinum Taq (Invitrogen Inc.). Fluorescence was
measured at 78-80.degree. C. during 40 cycles. Sunflower total RNA
was also prepared with the TRIZOL.RTM. (Invitrogen Inc.)
technique.
[0272] Chlorophyll Quantification
[0273] Extracts from 100 weighed mg of leaves were prepared after
freezing with liquid nitrogen. 1.5 ml of 80% acetone was added to
each sample, and the tubes were placed in darkness for a period of
30 minutes. During this incubation, the sample solids were decanted
and the absorbance at 645 and 663 nm, respectively, was measured in
the supernatants with a spectrophotometer. Chlorophyll
concentrations were quantified according to the methods disclosed
in Whatley et al. (1963).
[0274] Injury Evaluation by the Ion Leakage Technique
[0275] The ion leakage technique was carried out essentially as
described by Sukumaran and Weiser (1972) with minor modifications.
The conductivity percentage was calculated as the ratio between
C2/C1, i.e., [L=(C1/C2)*100], and was used as the index of injury.
C1 represents the conductance after the treatment, and C2
represents the maximum conductance of each sample. L values higher
than 50% indicate a severe injury.
[0276] Water Loss Evaluation
[0277] Fully expanded leaves were detached from Wild-Type (WT) and
the 35S:HaWRKY76 transgenic plants, respectively, and placed on
Petri dishes under strictly controlled conditions. Water loss was
determined by weighing the leaves at the times indicated in the
Figures as compared with the initial weight. The values are
reported as a percentage of the initial weight.
Example 1
[0278] Seeds of Helianthus annuus L. (sunflower CF31, from Advanta
Seeds) were grown in the soil pots in a culture chamber at
28.degree. C. for a period of 5 days. The HaWRKY76 transcription
levels were quantified by RT-PCR and normalized with housekeeping
ACTIN transcripts. The obtained values were normalized with respect
to the value measured in the root sample, which was arbitrarily
assigned a value of 1. An Anova test was performed, followed by a
Fisher LSD post-hoc test. The results are shown in FIG. 5. The
different letters (a and b) indicate samples that were found to be
significantly different (i.e., p value<0.05). As demonstrated by
the results summarized in FIG. 5, HaWRKY76 expression patterns in
5-day-old sunflower seedlings indicated higher levels in roots and
hypocotyls than in cotyledons.
Example 2
[0279] Seeds of Helianthus annuus L. (sunflower CF31, from Advanta
Seeds) were grown in the soil pots for 5 days under control
conditions and then placed for 30 minutes on a filter paper
(Drought) or in MS 0, 5.times. solution (Control). The HaWRKY76
transcription levels were quantified by RT-PCR and normalized with
housekeeping ACTIN transcripts. The obtained values were normalized
with respect to the value measured in the control root sample,
which was arbitrarily assigned a value of 1. An Anova test was
performed, followed by a Fisher LSD post-hoc test. The results are
shown in FIG. 6A. The different letters (a, b, and c) indicate
samples that were found to be significantly different (i.e., p
value<0.05).
[0280] Sunflower R2 plants were subjected to a continuous and
severe drought stress (stopping watering during 15 days). The leaf
disks were frozen at the period of time indicated in FIG. 6B. As a
control of the plants' viability, the plants were re-watered after
sampling. The transcription levels of HaWRKY76 were quantified in
these sunflower R2 plants subjected to a continuous and severe
drought stress. The obtained values were normalized with respect to
the value measured in the sample harvested at day 2 which was
arbitrarily assigned a value of 1. An Anova test was performed,
followed by a Fisher LSD post-hoc test. The results are shown in
FIG. 6B. As demonstrated by the results summarized in FIG. 6,
HaWRKY76 expression is up-regulated by water stress.
Example 3
[0281] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. Transcription levels of HaWRKY76 were quantified
by real time RT-PCR. All of the values were normalized with respect
to the value measured in line W76-C, which was arbitrarily assigned
a value of 1 and considered as a low-level-expression line. WT
plants were used as negative controls, and error bars correspond to
the standard deviations from three biological replicas. An Anova
test was performed, followed by a Fisher LSD post-hoc test. FIG. 7
shows the relative expression levels of HaWRKY76 in Arabidopsis
transgenic plants bearing the construct 35S:HaWRKY76. Different
letters (a, b, and c) indicate samples that were found to be
significantly different (i.e., p value<0.05).
Example 4
[0282] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The plants were grown in standard growth
conditions. The root length was measured in the 35S:HaWRKY76
(W76-A, W76-B and W76-C) and the WT plants. An Anova test was
performed, followed by a Fisher LSD post-hoc test. Error bars
correspond to the standard deviations twenty biological
replicas.
[0283] The average root lengths and standard deviations were
calculated in 7- and 14-day-old seedlings. The averages of each
genotype were calculated, and the results are shown in panel (A) of
FIG. 8. Different letters (a, b, c, and d) indicate samples that
were found to be significantly different (i.e., p value<0.05).
Panel (B) of FIG. 8 is a photograph of the roots of the 7-day-old
plants grown on Petri dishes. As demonstrated by the results shown
in FIG. 8, Arabidopsis plants bearing the construct 35S:HaWRKY76
develop longer roots than the control plants in standard growth
conditions.
Example 5
[0284] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. Roots were detached from 33-day-old 35S:HaWRKY76
(W76-A, W76-B and W76-C) and WT control plants grown on sand in
standard conditions, and the average weights of the respective
roots were measured. An Anova test was performed, followed by a
Fisher LSD post-hoc test. Error bars correspond to the standard
deviations from three biological replicas.
[0285] The average root weight of each genotype is shown in FIG. 9.
Different letters (a and b) indicate samples which were found to be
significantly different (i.e., p value<0.05). As demonstrated by
the results shown in FIG. 9, Arabidopsis plants bearing the
construct 35S:HaWRKY76 grown on sand in standard growth conditions
exhibit higher root biomass compared with WT control plants.
Example 6
[0286] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The plants were grown on poor soil in standard
growing conditions. Rosettes of 36-day-old 35S:HaWRKY76 (W76-A,
W76-B and W76-C) and WT plants were detached, and the weight of the
rosettes was measured. An Anova test was performed, followed by a
Fisher LSD post-hoc test. Error bars correspond to the standard
deviations from four or five biological replicas. The rosette
weight (mg) of each genotype is shown in panel (A) of FIG. 10.
[0287] The weighed leaves of the 36-day-old plants were frozen, and
chlorophyll was extracted with acetone and quantified by absorbance
at 645 and 663 nm, respectively (see "Chlorophyll Quantification"
conditions above). The total chlorophyll content per plant is shown
in panel (B) of FIG. 10. The protein content was also determined
using BSA as standard according to the method described in Bradford
(1976). The protein concentration per plant is shown in panel (C)
of FIG. 10.
[0288] As demonstrated by the results shown in FIG. 10, Arabidopsis
plants bearing the construct 35S:HaWRKY76 grown on poor soil in
standard conditions exhibit higher rosette biomass compared with
the WT control plants. Different letters (a and b) indicate samples
which were found to be significantly different (i.e., p
value<0.05). As also shown in FIG. 10B and FIG. 10C, the
chlorophyll content (.mu.g) per mg leaves is similar when comparing
genotypes. However, because rosettes of the transgenic plants are
larger, the total protein and chlorophyll contents differ when
comparing genotypes, i.e., the contents are larger in the
transgenic plants than in the WT control plants.
[0289] As further shown in FIG. 11, 35-day-old Arabidopsis plants
bearing the construct 35S:HaWRKY76 grown in standard conditions
exhibit higher aerial biomass compared with the WT control plants.
Error bars correspond to the standard deviations from four
biological replicas.
Example 7
[0290] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The plants were grown on poor soil in standard
growing conditions. The seeds of each plant from each of the
35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT genotype were
separately harvested and weighed. The average yield of 4 plants of
each genotype is shown in FIG. 12A. An Anova test was performed,
followed by a Fisher LSD post-hoc test. Different letters (a and b)
indicate samples which were found to be significantly different
(i.e., p value<0.05).
[0291] FIG. 12B shows the yield per plant, with the horizontal line
representing the average value of the WT plants. FIG. 12C is a
photograph of the harvested seeds of 4 plants of each genotype. As
demonstrated by the results shown in FIG. 12, Arabidopsis plants
bearing the construct 35S:HaWRKY76 grown in poor soil conditions
exhibit higher yields than the WT control plants.
[0292] As additionally shown in FIG. 13, Arabidopsis plants bearing
the construct 35S:HaWRKY76 grown on standard conditions also
exhibit higher yields than the WT control plants. Specifically,
FIG. 13 shows the average yield of 4 plants of each genotype. An
Anova test was performed, followed by a Fisher LSD post-hoc test.
Different letters (a and b) indicate samples which were found to be
significantly different (i.e., p value<0.05).
Example 8
[0293] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were well irrigated for 25 days. Then, the 25-day-old
well-irrigated plants were subjected to drought stress by stopping
watering until severe damage was visible. At that time, the plants
were re-watered. FIG. 14 is a photograph of the plants taken 4 days
after the re-watering.
[0294] As can be seen in the photograph of FIG. 14, Arabidopsis
plants bearing the construct 35S:HaWRKY76 are more tolerant to
drought than their WT control plants.
Example 9
[0295] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were grown on poor soil (undefined stress), and were
subjected to drought during the vegetative stage and also during
the reproductive stage. The plants were subjected to drought stress
by stopping watering until severe damage was visible. At that time,
the plants were re-watered.
[0296] FIG. 15A is a photograph of the plants subjected to drought
during the vegetative stage, and FIG. 15C is a photograph of the
plants subjected to drought during the reproductive stage.
[0297] The yield (seed production) of the plants was measured.
Error bars correspond to the standard deviations from four
biological replicas. An Anova test was performed, followed by a
Fisher LSC post-hoc test. FIG. 15B shows the yield of the plants
when subjected to drought during the vegetative stage, and FIG. 15D
shows the yield of the plants when subjected to drought during the
reproductive stage. Different letters (a and b) indicate samples
that were found to be significantly different (i.e., p
value<0.05).
[0298] As demonstrated by the results shown in FIG. 15, Arabidopsis
plants bearing the construct 35S:HaWRKY76 grown in poor soil
conditions are more tolerant to drought than their WT control
plants and show enhanced yield when the stress is applied during
the vegetative stage. When the stress was suffered during the
reproductive stage, no significant differences between the
genotypes were observed.
[0299] The top panel of FIG. 16 shows the enhanced yield of the
transgenic plants when the drought stress was suffered during the
vegetative stage, while the lower panel shows that no significant
differences in yield between the genotypes were observed when the
drought stress was suffered during the reproductive stage. In FIG.
16, error bars correspond to the standard deviations from four
biological replicas.
[0300] Similar experiments were conducted in a second set of
experiments and the results are shown in FIG. 35.
Example 10
[0301] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W6-B and W76-C) and WT
plants were grown on soil and normal watering stopped when the
plants were 25-days old (mild drought stress). Soil pots (poor
soil) were weighed every two days during the assay and water was
added to those that needed it to maintain the same weight in all
pots. After harvesting, plant yield was evaluated for each
individual plant of each genotype.
[0302] FIG. 17A shows the average amount (ml) of water added to the
soil for each genotype, and FIG. 17B shows the average yield per
plant of each genotype. An Anova test was performed, followed by a
Fisher LSC post-hoc test. Error bars correspond to the standard
deviations from 10 biological replicas. Different letters (a and b)
indicate samples that were found to be significantly different
(i.e., p value<0.05).
[0303] As demonstrated by the results shown in FIG. 17, Arabidopsis
plants bearing the construct 35S:HaWRKY76 need less water than the
WT control plants during a mild drought stress exhibiting similar
yields.
Example 11
[0304] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were well irrigated (FIG. 18A) or subjected to moderate
drought stress (FIG. 18B). In both cases, fully expanded leaves
were detached from the 35S:HaWRKY76 and the WT plants that were (A)
well irrigated and (B) subjected to moderate drought stress. The
detached leaves were placed on Petri dishes under controlled
conditions and weighed at the times indicated in FIGS. 18A and 18B,
respectively. The values are reported as weight at each measured
time with respect to the initial weight.
[0305] FIG. 18A shows the weight of leaves (% of initial weight) of
the well-irrigated plants, FIG. 18B shows the weight of leaves (%
of initial weight) of the plants subjected to moderate drought
stress, and FIG. 18C is a photograph taken after 8 hours of
treatment. An Anova test was performed, followed by a Fisher LSC
post-hoc test. Error bars correspond to the standard deviations
from 3 or 4 biological replicas. Different letters (a and b)
indicate samples that were found to be significantly different
(i.e., p value<0.05).
[0306] As demonstrated by the results shown in FIG. 18, Arabidopsis
plants bearing the construct 35S:HaWRKY76 lost less water than
their WT control plants.
Example 12
[0307] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were grown on poor soil for 25 days. The 25-day-old plants
were then completely submerged during 5-6 days and placed in
standard growing conditions until recovery. The number of survivors
after treatment and the average seed yield of the survivors were
calculated. One 6-day submergence experiment was performed with 12
plants of each genotype. As the number of survivors was not equal
for all genotypes, the seed yield of survivors was calculated from
one 5-day submerge experiment performed with 8 plants of each
genotype.
[0308] FIG. 19A shows the percentage of survivors of each genotype
after the treatment. FIG. 19B is a photograph of the plants taken
after one week of recovery. FIG. 19C shows the average seed yield
of survivors. An Anova test was performed, followed by a Fisher LSC
post-hoc test. Error bars correspond to the standard deviations
from 4-9 biological replicas. Different letters (a and b) indicate
samples which were found to be significantly different (i.e., p
value<0.05). FIG. 19D is an image of seeds produced by each
genotype with respect to the average seed yields shown in FIG.
19C.
[0309] As demonstrated by the results shown in FIG. 19, Arabidopsis
plants bearing the construct 35S:HaWRKY76 are more tolerant to
submergence and exhibited higher yields after such submergence than
their WT control plants.
[0310] The 25-day-old 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants subjected to submergence during 5-6 days as described above
were further evaluated according to different parameters after
recovery. Specifically, the rosette weight of the respective plants
was measured 2 and 5 days after the start of treatment
(submergence), and the results are shown in FIG. 20A. FIG. 20B is a
photograph of the rosettes taken 5 days after the start of
treatment. The chlorophyll content of the plants was also measured
at 2 and 5 days after the start of treatment, and the results are
shown in FIG. 20C. Finally, the total content of soluble sugars
(.mu.g/mg leaves) was determined for each of the plants, and the
results are shown in FIG. 20D. With respect to the results shown in
FIGS. 20A, 20C, and 20D, an Anova test was performed, followed by a
Fisher LSD post-hoc test. Error bars correspond to the standard
deviations from four biological replicas. Different letters
indicate samples that were found to be significantly different
(i.e., p value<0.05).
Example 13
[0311] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. Soluble carbohydrates and starch were
enzymatically determined in the rosettes of 25-day-old 35S:HaWRKY76
and WT plants that had been submerged during 2, 3, or 5 days.
[0312] FIG. 21A shows the soluble glucose per mg of fresh rosette
weight in for each of the plants. FIG. 21B shows the sucrose
content in the respective plants, and FIG. 21C shows the starch
content in the respective plants. As can be seen from the results
of FIG. 21, the transgenic plants have more soluble carbohydrates
during submergence than the WT control plants. Error bars
correspond to the standard deviations from 4 biological replicas.
An Anova test was performed, followed by a Fisher LSC post-hoc
test. Different letters (a, b, c, and d) indicate samples that were
found to be significantly different (i.e., p value<0.05).
Example 14
[0313] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. 25-day-old transgenic plants bearing the
construct 35S:HaWRKY76 and WT plants were completely submerged
during 5 days.
[0314] FIG. 22A shows the protein content per mg of fresh rosette
weight of the respective plants, and FIG. 22B shows the chlorophyll
content per mg of fresh rosette weight of the respective plants. As
can be seen from the quantified results in FIG. 22, the transgenic
plants exhibit higher protein concentrations than the WT control
plants during submergence. Error bars correspond to the standard
deviations from 4 biological replicas. An Anova test was performed,
followed by a Fisher LSC post-hoc test. Different letters (a, b, c,
and d) indicate samples that were found to be significantly
different (i.e., p value<0.05).
Example 15
[0315] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 transgenic plants and WT plants
were grown on sand for 35 days. On day 35, the complete root system
was collected and weighed.
[0316] FIG. 23A shows the root/aerial biomass ratio for each of the
respective plants, and FIG. 23B shows the protein content of the
roots collected from each of the respective plants. As can be seen
from the results in FIG. 23, the transgenic plants show a higher
root/aerial tissue biomass ratio than the WT control plants. Error
bars correspond to the standard deviations from 7 biological
replicas. An Anova test was performed, followed by a Fisher LSC
post-hoc test. Different letters (a, b) indicate samples that were
found to be significantly different (i.e., p value<0.05).
Example 16
[0317] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. 25-day-old 35S:HaWRKY76 transgenic plants and WT
plants were subjected to waterlogging during 5 days. Transverse
sections of stems of the respective plants were then obtained and
stained. The results are shown in the photographs of FIG. 24. As
can be seen from FIG. 24, the transgenic plants exhibit more
aerenchyma than the WT control plants.
Example 17
[0318] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were initially grown in standard growing conditions, then
subjected to submergence during 5 days, and finally recovered
during one additional day. The sucrose content of the respective
plants was measured.
[0319] FIG. 25 shows the sucrose content expressed as .mu.g of
glucose/mg of leaves, with each bar representing the average value
obtained from 4 plants. An Anova test was performed, followed by a
Fisher LSC post-hoc test. Error bars correspond to the standard
deviations from 4 biological replicas. Different letters (a, b, c,
and d) indicate samples that were found to be significantly
different (i.e., p value<0.05).
[0320] As demonstrated by the results shown in FIG. 25, the
transgenic plants exhibit higher sucrose content after submerge
than the WT control plants.
Example 18
[0321] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were initially grown in standard growing conditions.
Thereafter, 25-day-old plants of each genotype were completely
submerged during 5 days and placed in standard growing conditions
until recovery. The average yield was measured in 5 plants per
genotype.
[0322] The results are shown in FIG. 26. An Anova test was
performed, followed by a Fisher LSC post-hoc test. Error bars
correspond to the standard deviations. Different letters (a and b)
indicate samples that were found to be significantly different
(i.e., p value<0.05).
[0323] As demonstrated by the results shown in FIG. 26, the
transgenic plants exhibited slightly higher yields than the WT
control plants after 5 days of submergence.
Example 19
[0324] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were grown on soil for 25 days. Then, the 25-day-old plants
of each genotype were subjected to waterlogging treatment during
one week. The tolerance of the respective plants to the
waterlogging treatment was measured with respect to the rosette
weight, membrane stability, and stem length.
[0325] Panel (A) of FIG. 27 shows the rosette weights of the plants
measured 6 and 7 days after starting the waterlogging treatment.
Panel (B) of FIG. 27 shows the membrane stability (% of ion
leakage) measured 5 and 7 days after starting the treatment. Panel
(C) of FIG. 27 shows the stem lengths of the plants after the
waterlogging treatment, measured with a ruler. An Anova test was
performed, followed by a Fisher LSD post-hoc test. Error bars
correspond to standard deviations from 4 biological replicas.
Different letters indicate samples that were found to be
significantly different (i.e., p value<0.05).
[0326] As demonstrated by the results shown in FIG. 27, the
35S:HaWRKY76 transgenic plants are more tolerant to waterlogging
than the WT control plants.
Example 20
[0327] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were grown on poor soil for 25 days. Then, the 25-day-old
plants of each genotype were subjected to waterlogging treatment
during one week. The 35S:HaWRKY76 and WT plants' seeds were
harvested and weighed after the waterlogging treatment. All of the
plants survived the waterlogging treatment.
[0328] Panel (A) of FIG. 28 shows the average yield per genotype.
Panel (B) of FIG. 28 shows the yield of each plant, the horizontal
line representing the average value of the WT plants. An Anova test
was performed, followed by a Fisher LSD post-hoc test. Error bars
correspond to standard deviations from 4 biological replicas.
Different letters (a and b) indicate samples that were found to be
significantly different (i.e., p value<0.05).
[0329] As demonstrated by the results shown in FIG. 28, the
35S:HaWRKY76 transgenic plants exhibit higher yields after
waterlogging than the WT control plants.
Example 21
[0330] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were grown in standard soil, and then subjected to a
waterlogging treatment during one week. All of the plants survived
the waterlogging treatment.
[0331] Following the waterlogging treatment, seeds of the
35S:HaWRKY76 and WT plants were harvested and weighed. The top
panel of FIG. 29 shows the average yield per genotype, whereas the
bottom panel of FIG. 29 shows the yield of all the plants of each
genotype. An Anova test was performed, followed by a Fisher LSD
post-hoc test. Error bars correspond to standard deviations from 4
biological replicas. Different letters (a and b) indicate samples
that were found to be significantly different (i.e., p
value<0.05).
[0332] As demonstrated by the results shown in FIG. 29, the
35S:HaWRKY76 transgenic plants exhibit higher yields after a week
of waterlogging than the WT control plants.
Example 22
[0333] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were grown in standard growing conditions.
[0334] The number of rosette leaves of the 35S:HaWRKY76 and WT
plants was counted, and the results are shown in FIG. 30. Error
bars correspond to the standard deviations from eight biological
replicas. As demonstrated by the results shown in FIG. 30, when
grown in standard conditions, the transgenic plants do not exhibit
significant differences in the number of leaves/rosettes in
comparison to the WT control plants.
Example 23
[0335] Transgenic Arabidopsis thaliana plants bearing the construct
35S:HaWRKY76 were obtained by the floral dip method (see
"Constructs" above), and three homozygous lines were selected for
further analysis. The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT
plants were grown in standard growing conditions.
[0336] The stem lengths of the 35S:HaWRKY76 and WT control plants
was measured during their life cycle. Specifically, the stem
lengths were measured every 3-5 days starting during the plants'
transition from the vegetative to the reproductive stage. The
results are shown in FIG. 31. Error bars correspond to the standard
deviations from eight biological replicas. As demonstrated by the
results shown in FIG. 31, when grown in standard conditions, the
35S:HaWRKY76 transgenic plants do not exhibit a delay in their life
cycle in comparison to the WT control plants.
Example 24
[0337] FIG. 32 is an image of an EMSA assay performed with purified
recombinant HaWRKY76-GST. Lines 1 and 2 are 20 ng of W2-GST with a
12 N labeled nucleotide (10000 and 17000 cpm respectively). Line 4
is 20 ng of W2-GST with 10000 cpm of labeled oligonucleotide
exhibiting 2 W-Boxes (C/TTGACT/C) separated by 5 nucleotides. Lines
5 and 6 are negative controls without the protein and with the same
amount of labeled oligonucleotides used in lines 2 and 4
respectively.
[0338] The results of the assay show that the recombinant protein
HaWRKY76 binds with more affinity an oligonucleotide with a central
core 12 N (4 nucleotides per position) than a selected
oligonucleotide exhibiting two W-Boxes separated by 5 nucleotides.
This indicates a different binding affinity than their related WRKY
proteins.
INCORPORATION BY REFERENCE
[0339] All references and nucleotide and polypeptide sequences
cited are hereby incorporated by reference in their entireties
herein.
TABLE-US-00002 SEQUENCE INFORMATION HaWRK76 cDNA SEQ ID NO: 1
atggcggttgatttcgtcggaattcaatctaccgatcatcttctaaaccgcatgttccagtt
attaagtcacgatttaaacgtttcgtcaacctacacgcacgcggtttctgctttcaaacgca
ccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttca
acttcttcacattcggaaggtaaatcacgagatacgacttcgtttgtacaaaacgagtgttt
ttcaaacaaaccggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtag
tatcgtcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagttttcttcg
ttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacaca
ccggaaaaggtgcggcgctgatcgtcctgttgcttccgtacacggatccggaagcggttgcc
attgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagttccgattacc
ggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaa
gatcgacggttcactttatccacgagtatattacaaatgtattaccggaaaaggatgtccgg
cgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacgga
gaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtga
gccaaagtgaa HaWRK76 protein SEQ ID NO: 2
MAVDFVGIQSTDHLLNRMFQLLSHDLNVSSTYTHAVSAFKRTGHARFRRGPSSTTGDTNGPS
TSSHSEGKSRDTTSFVQNECFSNKPVTEITTTTTSTSSSSVVSSSTGGNLDGSVSNGKQFSS
LGIVAPAPTFSSRKPPLPSTHRKRCGADRPVASVHGSGSGCHCCSKRRKTGSKREIRRVPIT
GSKITSIPADDYSWKKYGEKKIDGSLYPRVYYKCITGKGCPARKRVELSADDSKMLIVTYDG
EHRHRDRHAPVPMSLIGVYGEPK HaT131007971 GENE SEQ ID NO: 3
ccccatctacccctcatatctctcatcaatctctctttctctctcttagggtttcaacttccaaccttcttcta-
caacaatggcggttgatttcgt
cggaattcaatctaccgatcatcttctaaaccgcatgttccagttattaagtcacgatttaaacgtttcgtcaa-
cctacacgcacgcggtttc
tgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggacctt-
caacttcttcacatt
cggaaggtaaatcacgagatacgacttcgtttgtacaaaacgagtgtttttcaaacaaatcggtgacggagata-
acgacgacgacgac
gtcaacgagctcgtcgtctgtagtatcttcttccaccggtggaaacttagacggaagtgtttccaacggtaaac-
agttttcttcgttaggta
tagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacacaccggaaaaggtgcggcgct-
gatcgtcctgttgc
ttccgtacacggatccggaagcggttgccattgttgttccaagagaaggaaaaccggatctaaacgtgaaatta-
gaagagttccgatta
ccggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggt-
tcactttatccacga
gtatattacaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaa-
gatgcttattgttac
ttacgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtgagt-
caaagtgaggggg
acacatgtgtggtccgtgagcactttgcacagttttctaaggtcaacaggaagagagagaaaataacttttttt-
attcttggtttagttgagg
gttaatttgtacatttgacaaaagatgaagggtgtaattggtaatttagaagatgcccccagatctgatattcg-
attttgtttggactaattact
ttataaaagttgatattggtatatttaaaatttaattaaagaggaaaagtaattagtccaaacaaaatcgaata-
tcagatctg HaT131007971 PROTEIN SEQ ID NO: 4 Met Ala Val Asp Phe Val
Gly Ile Gin Ser Thr Asp His Leu Leu Asn Arg Met Phe Gln Leu Leu Ser
His Asp Leu Asn Val Ser Ser Thr Tyr Thr His Ala Val Ser Ala Phe Lys
Arg Thr Gly Hi's Ala Arg Phe Arg Arg Gly Pro Ser Ser Thr Thr Gly
Asp Thr Asn Gly Pro Ser Thr Ser Ser His Ser Glu Gly Lys Ser Arg Asp
Thr Thr Ser Phe Val Gin Asn Glu Cys Phe Ser Asn Lys Ser Val Thr Glu
Ile Thr Thr Thr Thr Thr Ser Thr Ser Ser Ser Ser Val Val Ser Ser Ser
Thr Gly Gly Asn Leu Asp Gly Ser Val Ser Asn Gly Lys Gln Phe Ser Ser
Leu Gly Ile Val Ala Pro Ala Pro Thr Phe Ser Ser Arg Lys Pro Pro Leu
Pro Ser Thr His Arg Lys Arg Cys Gly Ala Asp Arg Pro Val Ala Ser Val
His Gly Ser Gly Ser Gly Cys His Cys Cys Ser Lys Arg Arg Lys Thr Gly
Ser Lys Arg Glu Ile Arg Arg Val Pro Ile Thr Gly Ser Lys Ile Thr Ser
Ile Pro Ala Asp Asp Tyr Ser Trp Lys Lys Tyr Gly Glu Lys Lys Ile Asp
Gly Ser Leu Tyr Pro Arg Val Tyr Tyr Lys Cys Ile Thr Gly Lys Gly Cys
Pro Ala Arg Lys Arg Val Glu Leu Ser Ala Asp Asp Ser Lys Met Leu Ile
Val Thr Tyr Asp Gly Glu His Arg His Arg Asp Arg His Ala Pro Val Pro
Met Ser Leu Thr Gly Val Tyr Gly Glu Ser Lys HuCL13748C001 GENE SEQ
ID NO: 5
cttggcggggatcatctctctttctctctctctctcttagggtttcaacttccaaccttcttctacaacaatgg-
cggttgatttcgtcggaattc
aatctacagatcatcatctaaaccgcatgtttcagttatcaactcacgatttaaacgtttcgtcaacctacaca-
cacgcggtttctgctttca
aacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttcaacttct-
tcacattcggaag
gtaaatcacgagatacgacgtcgtttgtacaaaacgagtgtttttcaaacaaatcggtgacggagataacgacg-
acgacgacgtcaac
gagctcgtcgtctgtagtatcgtcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagtttt-
tttcgttaggtatagtag
ctccggcgccgacgttctcgtctagaaaaccaccgctaccgtcgactcatcggaaaaggtgcagcgctgatcgt-
cctgttgcttccgta
cacggatctggaagcggttgccattgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagt-
tccgattaccggat
ctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggttcactt-
tatccacgagtgtatt
acaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgctt-
attgttacttacga
cggagaacaccgtcaccgtgaccgtcacgtgccggtacttatgagtttgaccggtgtgtatggtgagtcaaagt-
gagggggacacat gtgt HuCL13748C001 PROTEIN SEQ ID NO: 6 Met Ala Val
Asp Phe Val Gly Ile Gin Ser Thr Asp His His Leu Asn Arg Met Phe Gin
Leu Ser Thr His Asp Leu Asn Val Ser Ser Thr Tyr Thr His Ala Val Ser
Ala Phe Lys Arg Thr Gly His Ala Arg Phe Arg Arg Gly Pro Ser Ser Thr
Thr Gly Asp Thr Asn Gly Pro Ser Thr Ser Ser His Ser Glu Gly Lys Ser
Arg Asp Thr Thr Ser Phe Val Gln Asn Glu Cys Phe Ser Asn Lys Ser Val
Thr Glu Ile Thr Thr Thr Thr Thr Ser Thr Ser Ser Ser Ser Val Val Ser
Ser Ser Thr Gly Gly Asn Leu Asp Gly Ser Val Ser Asn Gly Lys Gin Phe
Phe Ser Leu Gly Ile Val Ala Pro Ala Pro Thr Phe Ser Ser Arg Lys Pro
Pro Leu Pro Ser Thr His Arg Lys Arg Cys Ser Ala Asp Arg Pro Val Ala
Ser Val His Gly Ser Gly Ser Gly Cys His Cys Cys Ser Lys Arg Arg Lys
Thr Gly Ser Lys Arg Glu Ile Arg Arg Val Pro Ile Thr Gly Ser Lys Ile
Thr Ser Ile Pro Ala Asp Asp Tyr Ser Trp Lys Lys Tyr Gly Glu Lys Lys
Ile Asp Gly Ser Leu Tyr Pro Arg Val Tyr Tyr Lys Cys Ile Thr Gly Lys
Gly Cys Pro Ala Arg Lys Arg Val Glu Leu Ser Ala Asp Asp Ser Lys Met
Leu Ile Val Thr Tyr Asp Gly Glu His Arg His Arg Asp Arg His Val Pro
Val Leu Met Ser Leu Thr Gly Val Tyr Gly Glu Ser Lys SEQ ID NO: 7
WKKYGEK SEQ ID NO: 8 WRKYGQK HaWRK76 genomic DNA SEQ ID NO: 9
aatacaattgattactcagtcgaataatggccaatatccgttcataatcgacgacgacgtca
ctagggttttttctcttccggttaccaggcctatcatctggtttctggtttattttgagggt
gagggttttgcattgattcctacgctccaatcatctgcttcctgaattgaatctgaatctga
atctgaaagttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagattcc
aagcattttatcgtgtgttgtaattttaatgaagcaatgaagattgaatatgcactagggtg
agggttttgcattgattcctgcgctccaatcatctgcttcctgaattgaatctgaatctgaa
agttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagaagagtatgttg
atataggagagttgaacccgacacgtcttctgtgatgattcaaaacattccgaataactagc
agaacccgatccacttacattcgaataacgctgagaatccacaacgcttatggccagattct
ttccttcaatcttggcaaaatcaatgcctgcttgttttattatcacgaaagtactgaacaac
cgagataggcacaataacaacaacagctttccatgatttggagactgatcggaatcgaatca
gtgattttactggaagccgtttgattatgttttattatcacgaaagtactgaacaaccgaga
tagacacaataacaacaacaactttccatgatttggagactgatcagaatcgaaacagtgat
tttactttactggaagccgtttgattatttcctcttggatctcgaaaggcacgttgtctgac
atcttgattttgattaccaactcaccatatcaatttctaggtagttaacaacgctaattaga
aaaaaaaacgtgattcatatacacaacggtctctttgcttcgtccactagaactatttgtgt
atgcattcttttggtagttaacaacgctaattagaaaaaaatctgtaaaattaatcatatcg
attgctataacaggatggatgtgacacacacagatagataagctgtcacagttgtcaagact
gtttgtcgggtttccaatgctcacacagggtgataaacaatgctgatacgaacacatgtgtt
tcgaaataggctttgttcttggtttaatgaagttacatgtaacatctacttatattcagatt
taaacaatgcccgcctcaactgagataacggacatccatcaagctcaaaaagaaaacttgtg
tttcccatagtcacaaaagattgcggtttgtatttcgcatagccaccgtttccgtagtaaaa
aacggttgcggtttgtatttcgcatagccaccgtttccgtagtaaaaaacggttgggtttgc
acattcaatcttcaatacttcattaaaataccactagtagaagaacccgacacgtcttcatg
ttacggagaatgttctacaaattgaaggaaaaggaaaagatttgttgaaatcgaatttgaca
actatgacagcttattaaaagtagcaatcacctccaatcatttcctggtcaaatcacacacc
ataaagtttaaagttatcgtgtgttgtagtttggaaagatgaccgaacaaagatctgaataa
caaactgtggagaacttgtttgtgtcgggttcttctgctagttacttgatcaacggactgat
cgttaacgttactctacttgtgctccgatttaagcggcgtgttccgatcaacgttgtttaaa
ggtgatgaagagtttagaaaccctagaatgatggtgcttttacgggaaaaaacgtgataatt
gaaaaacatcaatctatacggcctgtatacaattatacggcccgtatacatttggtaaacat
caaaaacctcccaaaataaatccattgagccgtatagtttttacaaatcgtatacacatgta
tacggctcgtataactatatacgggccgtatatagtaaaaaaaacattctttacgcattgtc
ctattaaaaaacattctatacggaccgtatattttgtatacggtccgtataaactaaagata
tgaaaataaaatatagggggtgtgggaataagaggagcgttattaaaatacacaaataattt
tcgtcatgactccaactacatgttttattgtgttcttgtttgtcgttattaatattttcttt
gacatagaaagtcgaaacgcatggtatccagctacgtacacatttattcaacttatatttat
tttagtcctcgtttgactttatgcctccactagctttctccatctgtttatcacgctgcaaa
taagtcaaacaattctccaccgtccgatcaaaacagtatctcattgactccactttcccaat
tggacctttataacccccttctacccctcatatctctcatcaatctctctttctctctctta
gggtttcaacttccaaccttcttctacaacaatggcggttgatttcgtcggaattcaatcta
ccgatcatcttctaaaccgcatgttccagttattaagtcacgatttaaacgtttcgtcaacc
tacacgcacgcggtttctgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtc
gtctaccaccggagacactaacggaccttcaacttcttcacattcggaaggtaaatcacgag
atacgacttcgtttgtacaaaacgagtgtttttcaaacaaatcggtgacggagataacgacg
acgacgacgtcaacgagctcgtcgtctgtagtatcttcttccaccggtggaaacttagacgg
aagtgtttccaacggtaaacagttttcttcgttaggtatagtagctccggcgccgacgttct
cgtctagaaaaccaccgttaccgtcgacacaccggaaaaggtgcggcgctgatcgtcctgtt
gcttccgtacacggatccggaagcggttgccattgggaaaaccggatctaaacgtgaaatta
gaagagttccgattaccggatctaaaattacaagcatacctgctgatgattactcatggaaa
aagtacggcgagaagaagatcgacggttcactttatccacgagtatattacaaatgtattac
cggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgctta
ttgttacttacgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttg
accggtgtgtatggtgagtcaaagtgagggggacacatgtgtggtccgtgagcactttgcac
agttttctaaggtcaacaggaagagagagaaattaactttttttattcttggtttagttgag
ggttaatttgtacatttgacaaaagatgaagggtgtaattggtaatttagaagatgccccag
atctgatattcgattttgtttggactaattactttataaaagttgatattggtatatttaaa
atttaattaaagaggaaaagtaatttgaataagtttgatgccacagcagagtcaatgggttt
taaagtctctttaaatgactaaaaaattagataattgaatgaattttttaatggcaaatgta
gtctttattttcatatttattatggtcgttggtgcgacttttggcaaattgatttcgacaat
gtattgatggcgatgatctggaatggtccaatccaattttttatttgttttattgtttaata
tttaggagactttggaaaaatagcaagggttgaccctgatgaatattaataagttgtgttta
ctaaagaagcaagaatgtaacagctagcgatgagatgttaactaacgggtaccgtattgatg
tcgagctaaaaccaaaaccaaataacataatgtgtatgttcagttggtattggtattatacg
gtaccgctaaaaatcccaaacaggtaagtgccacatggtatcgatactgcagcttagataaa
ataaaattgaatatcacaccgtatatgtttggtcgacatcaataacaggtattcggtatgaa
gtttcccatctcaagactggcacgtaatatcatatatatacaactatagttggttttatact
ttcggagtttacggtttcaacttctattagttgggtgaagagcatccaagaggtcatcaatc tgta
HaWRK76 promoter 5'UTRs underlined SEQ ID NO: 10
aatacaattgattactcagtcgaataatggccaatatccgttcataatcgacgacgacgtca
ctagggttttttctcttccggttaccaggcctatcatctggtttctggtttattttgagggt
gagggttttgcattgattcctacgctccaatcatctgcttcctgaattgaatctgaatctga
atctgaaagttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagattcc
aagcattttatcgtgtgttgtaattttaatgaagcaatgaagattgaatatgcactagggtg
agggttttgcattgattcctgcgctccaatcatctgcttcctgaattgaatctgaatctgaa
agttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagaagagtatgttg
atataggagagttgaacccgacacgtcttctgtgatgattcaaaacattccgaataactagc
agaacccgatccacttacattcgaataacgctgagaatccacaacgcttatggccagattct
ttccttcaatcttggcaaaatcaatgcctgcttgttttattatcacgaaagtactgaacaac
cgagataggcacaataacaacaacagctttccatgatttggagactgatcggaatcgaatca
gtgattttactggaagccgtttgattatgttttattatcacgaaagtactgaacaaccgaga
tagacacaataacaacaacaactttccatgatttggagactgatcagaatcgaaacagtgat
tttactttactggaagccgtttgattatttcctcttggatctcgaaaggcacgttgtctgac
atcttgattttgattaccaactcaccatatcaatttctaggtagttaacaacgctaattaga
aaaaaaaacgtgattcatatacacaacggtctctttgcttcgtccactagaactatttgtgt
atgcattcttttggtagttaacaacgctaattagaaaaaaatctgtaaaattaatcatatcg
attgctataacaggatggatgtgacacacacagatagataagctgtcacagttgtcaagact
gtttgtcgggtttccaatgctcacacagggtgataaacaatgctgatacgaacacatgtgtt
tcgaaataggctttgttcttggtttaatgaagttacatgtaacatctacttatattcagatt
taaacaatgcccgcctcaactgagataacggacatccatcaagctcaaaaagaaaacttgtg
tttcccatagtcacaaaagattgcggtttgtatttcgcatagccaccgtttccgtagtaaaa
aacggttgcggtttgtatttcgcatagccaccgtttccgtagtaaaaaacggttgggtttgc
acattcaatcttcaatacttcattaaaataccactagtagaagaacccgacacgtcttcatg
ttacggagaatgttctacaaattgaaggaaaaggaaaagatttgttgaaatcgaatttgaca
actatgacagcttattaaaagtagcaatcacctccaatcatttcctggtcaaatcacacacc
ataaagtttaaagttatcgtgtgttgtagtttggaaagatgaccgaacaaagatctgaataa
caaactgtggagaacttgtttgtgtcgggttcttctgctagttacttgatcaacggactgat
cgttaacgttactctacttgtgctccgatttaagcggcgtgttccgatcaacgttgtttaaa
ggtgatgaagagtttagaaaccctagaatgatggtgcttttacgggaaaaaacgtgataatt
gaaaaacatcaatctatacggcctgtatacaattatacggcccgtatacatttggtaaacat
caaaaacctcccaaaataaatccattgagccgtatagtttttacaaatcgtatacacatgta
tacggctcgtataactatatacgggccgtatatagtaaaaaaaacattctttacgcattgtc
ctattaaaaaacattctatacggaccgtatattttgtatacggtccgtataaactaaagata
tgaaaataaaatatagggggtgtgggaataagaggagcgttattaaaatacacaaataattt
tcgtcatgactccaactacatgttttattgtgttcttgtttgtcgttattaatattttcttt
gacatagaaagtcgaaacgcatggtatccagctacgtacacatttattcaacttatatttat
tttagtcctcgtttgactttatgcctccactagctttctccatctgtttatcacgctgcaaa
taagtcaaacaattctccaccgtccgatcaaaacagtatctcattgactccactttcccaat
tggacctttataacccccttctacccctcatatctctcatcaatctctctttctctctctta
gggtttcaacttccaaccttcttctacaaca variant HaWRK76 cDNA. This has a g
in position 220 (underlined) and encodes a protein as shown in SEQ
ID No. 12 has an A in the motif SRDAT instead of a T. SEQ ID NO: 11
Atggcggttgatttcgtcggaattcaatctaccgatcatcttctaaaccgcatgttccagtt
attaagtcacgatttaaacgtttcgtcaacctacacgcacgcggtttctgctttcaaacgca
ccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttca
acttcttcacattcggaaggtaaatcacgagatgcgacttcgtttgtacaaaacgagtgttt
ttcaaacaaaccggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtag
tatcgtcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagttttcttcg
ttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacaca
ccggaaaaggtgcggcgctgatcgtcctgttgcttccgtacacggatccggaagcggttgcc
attgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagttccgattacc
ggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaa
gatcgacggttcactttatccacgagtatattacaaatgtattaccggaaaaggatgtccgg
cgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacgga
gaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtga
gccaaagtgaa variant HaWRK76 protein. This has an A in the motif
SRDAT instead of a T (see underlined A) SEQ ID NO: 12
MAVDFVGIQSTDHLLNRMFQLLSHDLNVSSTYTHAVSAFKRTGHARFRRGPSSTTGDTNGPS
TSSHSEGKSRDATSFVQNECFSNKPVTEITTTTTSTSSSSVVSSSTGGNLDGSVSNGKQFSS
LGIVAPAPTFSSRKPPLPSTHRKRCGADRPVASVHGSGSGCHCCSKRRKTGSKREIRRVPIT
GSKITSIPADDYSWKKYGEKKIDGSLYPRVYYKCITGKGCPARKRVELSADDSKMLIVTYDG
EHRHRDRHAPVPMSLIGVYGEPK
Sequence CWU 1
1
121817DNAHelianthus annuus 1atggcggttg atttcgtcgg aattcaatct
accgatcatc ttctaaaccg catgttccag 60ttattaagtc acgatttaaa cgtttcgtca
acctacacgc acgcggtttc tgctttcaaa 120cgcaccggtc acgcacggtt
ccgccgtgga ccgtcgtcta ccaccggaga cactaacgga 180ccttcaactt
cttcacattc ggaaggtaaa tcacgagatg cgacttcgtt tgtacaaaac
240gagtgttttt caaacaaacc ggtgacggag ataacgacga cgacgacgtc
aacgagctcg 300tcgtctgtag tatcgtcttc caccggtgga aacttagacg
gaagtgtttc caacggtaaa 360cagttttctt cgttaggtat agtagctccg
gcgccgacgt tctcgtctag aaaaccaccg 420ttaccgtcga cacaccggaa
aaggtgcggc gctgatcgtc ctgttgcttc cgtacacgga 480tccggaagcg
gttgccattg ttgttccaag agaaggaaaa ccggatctaa acgtgaaatt
540agaagagttc cgattaccgg atctaaaatt acaagcatac ctgctgatga
ttactcatgg 600aaaaagtacg gcgagaagaa gatcgacggt tcactttatc
cacgagtata ttacaaatgt 660attaccggaa aaggatgtcc ggcgaggaag
cgcgtggagt taagcgccga cgattcgaag 720atgcttattg ttacttacga
cggagaacac cgtcaccgtg accgtcacgc gccggtacct 780atgagtttga
ccggtgtgta tggtgagcca aagtgaa 8172271PRTHelianthus annuus 2Met Ala
Val Asp Phe Val Gly Ile Gln Ser Thr Asp His Leu Leu Asn1 5 10 15Arg
Met Phe Gln Leu Leu Ser His Asp Leu Asn Val Ser Ser Thr Tyr 20 25
30Thr His Ala Val Ser Ala Phe Lys Arg Thr Gly His Ala Arg Phe Arg
35 40 45Arg Gly Pro Ser Ser Thr Thr Gly Asp Thr Asn Gly Pro Ser Thr
Ser 50 55 60Ser His Ser Glu Gly Lys Ser Arg Asp Thr Thr Ser Phe Val
Gln Asn65 70 75 80Glu Cys Phe Ser Asn Lys Pro Val Thr Glu Ile Thr
Thr Thr Thr Thr 85 90 95Ser Thr Ser Ser Ser Ser Val Val Ser Ser Ser
Thr Gly Gly Asn Leu 100 105 110Asp Gly Ser Val Ser Asn Gly Lys Gln
Phe Ser Ser Leu Gly Ile Val 115 120 125Ala Pro Ala Pro Thr Phe Ser
Ser Arg Lys Pro Pro Leu Pro Ser Thr 130 135 140His Arg Lys Arg Cys
Gly Ala Asp Arg Pro Val Ala Ser Val His Gly145 150 155 160Ser Gly
Ser Gly Cys His Cys Cys Ser Lys Arg Arg Lys Thr Gly Ser 165 170
175Lys Arg Glu Ile Arg Arg Val Pro Ile Thr Gly Ser Lys Ile Thr Ser
180 185 190Ile Pro Ala Asp Asp Tyr Ser Trp Lys Lys Tyr Gly Glu Lys
Lys Ile 195 200 205Asp Gly Ser Leu Tyr Pro Arg Val Tyr Tyr Lys Cys
Ile Thr Gly Lys 210 215 220Gly Cys Pro Ala Arg Lys Arg Val Glu Leu
Ser Ala Asp Asp Ser Lys225 230 235 240Met Leu Ile Val Thr Tyr Asp
Gly Glu His Arg His Arg Asp Arg His 245 250 255Ala Pro Val Pro Met
Ser Leu Thr Gly Val Tyr Gly Glu Pro Lys 260 265
27031172DNAHelianthus annuus 3cccccttcta cccctcatat ctctcatcaa
tctctctttc tctctcttag ggtttcaact 60tccaaccttc ttctacaaca atggcggttg
atttcgtcgg aattcaatct accgatcatc 120ttctaaaccg catgttccag
ttattaagtc acgatttaaa cgtttcgtca acctacacgc 180acgcggtttc
tgctttcaaa cgcaccggtc acgcacggtt ccgccgtgga ccgtcgtcta
240ccaccggaga cactaacgga ccttcaactt cttcacattc ggaaggtaaa
tcacgagata 300cgacttcgtt tgtacaaaac gagtgttttt caaacaaatc
ggtgacggag ataacgacga 360cgacgacgtc aacgagctcg tcgtctgtag
tatcttcttc caccggtgga aacttagacg 420gaagtgtttc caacggtaaa
cagttttctt cgttaggtat agtagctccg gcgccgacgt 480tctcgtctag
aaaaccaccg ttaccgtcga cacaccggaa aaggtgcggc gctgatcgtc
540ctgttgcttc cgtacacgga tccggaagcg gttgccattg ttgttccaag
agaaggaaaa 600ccggatctaa acgtgaaatt agaagagttc cgattaccgg
atctaaaatt acaagcatac 660ctgctgatga ttactcatgg aaaaagtacg
gcgagaagaa gatcgacggt tcactttatc 720cacgagtata ttacaaatgt
attaccggaa aaggatgtcc ggcgaggaag cgcgtggagt 780taagcgccga
cgattcgaag atgcttattg ttacttacga cggagaacac cgtcaccgtg
840accgtcacgc gccggtacct atgagtttga ccggtgtgta tggtgagtca
aagtgagggg 900gacacatgtg tggtccgtga gcactttgca cagttttcta
aggtcaacag gaagagagag 960aaaataactt tttttattct tggtttagtt
gagggttaat ttgtacattt gacaaaagat 1020gaagggtgta attggtaatt
tagaagatgc ccccagatct gatattcgat tttgtttgga 1080ctaattactt
tataaaagtt gatattggta tatttaaaat ttaattaaag aggaaaagta
1140attagtccaa acaaaatcga atatcagatc tg 11724267PRTHelianthus
annuus 4Met Ala Val Asp Phe Val Gly Ile Ser Thr Asp His Leu Leu Asn
Arg1 5 10 15Met Phe Gln Leu Leu Ser His Asp Leu Asn Val Ser Ser Thr
Tyr Thr 20 25 30His Ala Val Ser Ala Phe Lys Arg Thr Gly Ala Arg Phe
Arg Arg Gly 35 40 45Pro Ser Ser Thr Thr Gly Asp Thr Asn Gly Pro Ser
Thr Ser Ser His 50 55 60Ser Glu Gly Lys Ser Arg Asp Thr Thr Ser Phe
Val Asn Glu Cys Phe65 70 75 80Ser Asn Lys Ser Val Thr Glu Ile Thr
Thr Thr Thr Thr Ser Thr Ser 85 90 95Ser Ser Ser Val Val Ser Ser Ser
Thr Gly Gly Asn Leu Asp Gly Ser 100 105 110Val Ser Asn Gly Lys Phe
Ser Ser Leu Gly Ile Val Ala Pro Ala Pro 115 120 125Thr Phe Ser Ser
Arg Lys Pro Pro Leu Pro Ser Thr His Arg Lys Arg 130 135 140Cys Gly
Ala Asp Arg Pro Val Ala Ser Val His Gly Ser Gly Ser Gly145 150 155
160Cys His Cys Cys Ser Lys Arg Arg Lys Thr Gly Ser Lys Arg Glu Ile
165 170 175Arg Arg Val Pro Ile Thr Gly Ser Lys Ile Thr Ser Ile Pro
Ala Asp 180 185 190Asp Tyr Ser Trp Lys Lys Tyr Gly Glu Lys Lys Ile
Asp Gly Ser Leu 195 200 205Tyr Pro Arg Val Tyr Tyr Lys Cys Ile Thr
Gly Lys Gly Cys Pro Ala 210 215 220Arg Lys Arg Val Glu Leu Ser Ala
Asp Asp Ser Lys Met Leu Ile Val225 230 235 240Thr Tyr Asp Gly Glu
His Arg His Arg Asp Arg His Ala Pro Val Pro 245 250 255Met Ser Leu
Thr Gly Val Tyr Gly Glu Ser Lys 260 2655901DNAHelianthus annuus
5cttggcgggg atcatctctc tttctctctc tctctcttag ggtttcaact tccaaccttc
60ttctacaaca atggcggttg atttcgtcgg aattcaatct acagatcatc atctaaaccg
120catgtttcag ttatcaactc acgatttaaa cgtttcgtca acctacacac
acgcggtttc 180tgctttcaaa cgcaccggtc acgcacggtt ccgccgtgga
ccgtcgtcta ccaccggaga 240cactaacgga ccttcaactt cttcacattc
ggaaggtaaa tcacgagata cgacgtcgtt 300tgtacaaaac gagtgttttt
caaacaaatc ggtgacggag ataacgacga cgacgacgtc 360aacgagctcg
tcgtctgtag tatcgtcttc caccggtgga aacttagacg gaagtgtttc
420caacggtaaa cagttttttt cgttaggtat agtagctccg gcgccgacgt
tctcgtctag 480aaaaccaccg ctaccgtcga ctcatcggaa aaggtgcagc
gctgatcgtc ctgttgcttc 540cgtacacgga tctggaagcg gttgccattg
ttgttccaag agaaggaaaa ccggatctaa 600acgtgaaatt agaagagttc
cgattaccgg atctaaaatt acaagcatac ctgctgatga 660ttactcatgg
aaaaagtacg gcgagaagaa gatcgacggt tcactttatc cacgagtgta
720ttacaaatgt attaccggaa aaggatgtcc ggcgaggaag cgcgtggagt
taagcgccga 780cgattcgaag atgcttattg ttacttacga cggagaacac
cgtcaccgtg accgtcacgt 840gccggtactt atgagtttga ccggtgtgta
tggtgagtca aagtgagggg gacacatgtg 900t 9016267PRTHelianthus annuus
6Met Ala Val Asp Phe Val Gly Ile Ser Thr Asp His His Leu Asn Arg1 5
10 15Met Phe Leu Ser Thr His Asp Leu Asn Val Ser Ser Thr Tyr Thr
His 20 25 30Ala Val Ser Ala Phe Lys Arg Thr Gly His Ala Arg Phe Arg
Arg Gly 35 40 45Pro Ser Ser Thr Thr Gly Asp Thr Asn Gly Pro Ser Thr
Ser Ser His 50 55 60Ser Glu Gly Lys Ser Arg Asp Thr Thr Ser Phe Val
Asn Glu Cys Phe65 70 75 80Ser Asn Lys Ser Val Thr Glu Ile Thr Thr
Thr Thr Thr Ser Thr Ser 85 90 95Ser Ser Ser Val Val Ser Ser Ser Thr
Gly Gly Asn Leu Asp Gly Ser 100 105 110Val Ser Asn Gly Lys Phe Phe
Ser Leu Gly Ile Val Ala Pro Ala Pro 115 120 125Thr Phe Ser Ser Arg
Lys Pro Pro Leu Pro Ser Thr His Arg Lys Arg 130 135 140Cys Ser Ala
Asp Arg Pro Val Ala Ser Val His Gly Ser Gly Ser Gly145 150 155
160Cys His Cys Cys Ser Lys Arg Arg Lys Thr Gly Ser Lys Arg Glu Ile
165 170 175Arg Arg Val Pro Ile Thr Gly Ser Lys Ile Thr Ser Ile Pro
Ala Asp 180 185 190Asp Tyr Ser Trp Lys Lys Tyr Gly Glu Lys Lys Ile
Asp Gly Ser Leu 195 200 205Tyr Pro Arg Val Tyr Tyr Lys Cys Ile Thr
Gly Lys Gly Cys Pro Ala 210 215 220Arg Lys Arg Val Glu Leu Ser Ala
Asp Asp Ser Lys Met Leu Ile Val225 230 235 240Thr Tyr Asp Gly Glu
His Arg His Arg Asp Arg His Val Pro Val Leu 245 250 255Met Ser Leu
Thr Gly Val Tyr Gly Glu Ser Lys 260 26577PRTHelianthus annuus 7Trp
Lys Lys Tyr Gly Glu Lys1 587PRTHelianthus annuus 8Trp Arg Lys Tyr
Gly Gln Lys1 594454DNAHelianthus annuus 9aatacaattg attactcagt
cgaataatgg ccaatatccg ttcataatcg acgacgacgt 60cactagggtt ttttctcttc
cggttaccag gcctatcatc tggtttctgg tttattttga 120gggtgagggt
tttgcattga ttcctacgct ccaatcatct gcttcctgaa ttgaatctga
180atctgaatct gaaagttaac tagacccctc aagttgtcat tcgtaacaac
taagcgtctg 240gagattccaa gcattttatc gtgtgttgta attttaatga
agcaatgaag attgaatatg 300cactagggtg agggttttgc attgattcct
gcgctccaat catctgcttc ctgaattgaa 360tctgaatctg aaagttaact
agacccctca agttgtcatt cgtaacaact aagcgtctgg 420agaagagtat
gttgatatag gagagttgaa cccgacacgt cttctgtgat gattcaaaac
480attccgaata actagcagaa cccgatccac ttacattcga ataacgctga
gaatccacaa 540cgcttatggc cagattcttt ccttcaatct tggcaaaatc
aatgcctgct tgttttatta 600tcacgaaagt actgaacaac cgagataggc
acaataacaa caacagcttt ccatgatttg 660gagactgatc ggaatcgaat
cagtgatttt actggaagcc gtttgattat gttttattat 720cacgaaagta
ctgaacaacc gagatagaca caataacaac aacaactttc catgatttgg
780agactgatca gaatcgaaac agtgatttta ctttactgga agccgtttga
ttatttcctc 840ttggatctcg aaaggcacgt tgtctgacat cttgattttg
attaccaact caccatatca 900atttctaggt agttaacaac gctaattaga
aaaaaaaacg tgattcatat acacaacggt 960ctctttgctt cgtccactag
aactatttgt gtatgcattc ttttggtagt taacaacgct 1020aattagaaaa
aaatctgtaa aattaatcat atcgattgct ataacaggat ggatgtgaca
1080cacacagata gataagctgt cacagttgtc aagactgttt gtcgggtttc
caatgctcac 1140acagggtgat aaacaatgct gatacgaaca catgtgtttc
gaaataggct ttgttcttgg 1200tttaatgaag ttacatgtaa catctactta
tattcagatt taaacaatgc ccgcctcaac 1260tgagataacg gacatccatc
aagctcaaaa agaaaacttg tgtttcccat agtcacaaaa 1320gattgcggtt
tgtatttcgc atagccaccg tttccgtagt aaaaaacggt tgcggtttgt
1380atttcgcata gccaccgttt ccgtagtaaa aaacggttgg gtttgcacat
tcaatcttca 1440atacttcatt aaaataccac tagtagaaga acccgacacg
tcttcatgtt acggagaatg 1500ttctacaaat tgaaggaaaa ggaaaagatt
tgttgaaatc gaatttgaca actatgacag 1560cttattaaaa gtagcaatca
cctccaatca tttcctggtc aaatcacaca ccataaagtt 1620taaagttatc
gtgtgttgta gtttggaaag atgaccgaac aaagatctga ataacaaact
1680gtggagaact tgtttgtgtc gggttcttct gctagttact tgatcaacgg
actgatcgtt 1740aacgttactc tacttgtgct ccgatttaag cggcgtgttc
cgatcaacgt tgtttaaagg 1800tgatgaagag tttagaaacc ctagaatgat
ggtgctttta cgggaaaaaa cgtgataatt 1860gaaaaacatc aatctatacg
gcctgtatac aattatacgg cccgtataca tttggtaaac 1920atcaaaaacc
tcccaaaata aatccattga gccgtatagt ttttacaaat cgtatacaca
1980tgtatacggc tcgtataact atatacgggc cgtatatagt aaaaaaaaca
ttctttacgc 2040attgtcctat taaaaaacat tctatacgga ccgtatattt
tgtatacggt ccgtataaac 2100taaagatatg aaaataaaat atagggggtg
tgggaataag aggagcgtta ttaaaataca 2160caaataattt tcgtcatgac
tccaactaca tgttttattg tgttcttgtt tgtcgttatt 2220aatattttct
ttgacataga aagtcgaaac gcatggtatc cagctacgta cacatttatt
2280caacttatat ttattttagt cctcgtttga ctttatgcct ccactagctt
tctccatctg 2340tttatcacgc tgcaaataag tcaaacaatt ctccaccgtc
cgatcaaaac agtatctcat 2400tgactccact ttcccaattg gacctttata
acccccttct acccctcata tctctcatca 2460atctctcttt ctctctctta
gggtttcaac ttccaacctt cttctacaac aatggcggtt 2520gatttcgtcg
gaattcaatc taccgatcat cttctaaacc gcatgttcca gttattaagt
2580cacgatttaa acgtttcgtc aacctacacg cacgcggttt ctgctttcaa
acgcaccggt 2640cacgcacggt tccgccgtgg accgtcgtct accaccggag
acactaacgg accttcaact 2700tcttcacatt cggaaggtaa atcacgagat
acgacttcgt ttgtacaaaa cgagtgtttt 2760tcaaacaaat cggtgacgga
gataacgacg acgacgacgt caacgagctc gtcgtctgta 2820gtatcttctt
ccaccggtgg aaacttagac ggaagtgttt ccaacggtaa acagttttct
2880tcgttaggta tagtagctcc ggcgccgacg ttctcgtcta gaaaaccacc
gttaccgtcg 2940acacaccgga aaaggtgcgg cgctgatcgt cctgttgctt
ccgtacacgg atccggaagc 3000ggttgccatt gttgttccaa gagaaggtgc
gtatgcgctc tcggcgaacg ttagcaaatt 3060tatgtactta ttatatatct
tacgcctgtt ttcgtaaagt aaactaataa aaatatcttc 3120cttttcgtgt
attctctcag gaaaaccgga tctaaacgtg aaattagaag agttccgatt
3180accggatcta aaattacaag catacctgct gatgattact catggaaaaa
gtacggcgag 3240aagaagatcg acggttcact ttatccacgg taattaatca
cagtcttcat aaatttataa 3300tatttataat tataattata attataatta
taatttaatg gatttctgat ttagattcta 3360atttgaaata tacagagtat
attacaaatg tattaccgga aaaggatgtc cggcgaggaa 3420gcgcgtggag
ttaagcgccg acgattcgaa gatgcttatt gttacttacg acggagaaca
3480ccgtcaccgt gaccgtcacg cgccggtacc tatgagtttg accggtgtgt
atggtgagtc 3540aaagtgaggg ggacacatgt gtggtccgtg agcactttgc
acagttttct aaggtcaaca 3600ggaagagaga gaaattaact ttttttattc
ttggtttagt tgagggttaa tttgtacatt 3660tgacaaaaga tgaagggtgt
aattggtaat ttagaagatg ccccagatct gatattcgat 3720tttgtttgga
ctaattactt tataaaagtt gatattggta tatttaaaat ttaattaaag
3780aggaaaagta atttgaataa gtttgatgcc acagcagagt caatgggttt
taaagtctct 3840ttaaatgact aaaaaattag ataattgaat gaatttttta
atggcaaatg tagtctttat 3900tttcatattt attatggtcg ttggtgcgac
ttttggcaaa ttgatttcga caatgtattg 3960atggcgatga tctggaatgg
tccaatccaa ttttttattt gttttattgt ttaatattta 4020ggagactttg
gaaaaatagc aagggttgac cctgatgaat attaataagt tgtgtttact
4080aaagaagcaa gaatgtaaca gctagcgatg agatgttaac taacgggtac
cgtattgatg 4140tcgagctaaa accaaaacca aataacataa tgtgtatgtt
cagttggtat tggtattata 4200cggtaccgct aaaaatccca aacaggtaag
tgccacatgg tatcgatact gcagcttaga 4260taaaataaaa ttgaatatca
caccgtatat gtttggtcga catcaataac aggtattcgg 4320tatgaagttt
cccatctcaa gactggcacg taatatcata tatatacaac tatagttggt
4380tttatacttt cggagtttac ggtttcaact tctattagtt gggtgaagag
catccaagag 4440gtcatcaatc tgta 4454102511DNAHelianthus annuus
10aatacaattg attactcagt cgaataatgg ccaatatccg ttcataatcg acgacgacgt
60cactagggtt ttttctcttc cggttaccag gcctatcatc tggtttctgg tttattttga
120gggtgagggt tttgcattga ttcctacgct ccaatcatct gcttcctgaa
ttgaatctga 180atctgaatct gaaagttaac tagacccctc aagttgtcat
tcgtaacaac taagcgtctg 240gagattccaa gcattttatc gtgtgttgta
attttaatga agcaatgaag attgaatatg 300cactagggtg agggttttgc
attgattcct gcgctccaat catctgcttc ctgaattgaa 360tctgaatctg
aaagttaact agacccctca agttgtcatt cgtaacaact aagcgtctgg
420agaagagtat gttgatatag gagagttgaa cccgacacgt cttctgtgat
gattcaaaac 480attccgaata actagcagaa cccgatccac ttacattcga
ataacgctga gaatccacaa 540cgcttatggc cagattcttt ccttcaatct
tggcaaaatc aatgcctgct tgttttatta 600tcacgaaagt actgaacaac
cgagataggc acaataacaa caacagcttt ccatgatttg 660gagactgatc
ggaatcgaat cagtgatttt actggaagcc gtttgattat gttttattat
720cacgaaagta ctgaacaacc gagatagaca caataacaac aacaactttc
catgatttgg 780agactgatca gaatcgaaac agtgatttta ctttactgga
agccgtttga ttatttcctc 840ttggatctcg aaaggcacgt tgtctgacat
cttgattttg attaccaact caccatatca 900atttctaggt agttaacaac
gctaattaga aaaaaaaacg tgattcatat acacaacggt 960ctctttgctt
cgtccactag aactatttgt gtatgcattc ttttggtagt taacaacgct
1020aattagaaaa aaatctgtaa aattaatcat atcgattgct ataacaggat
ggatgtgaca 1080cacacagata gataagctgt cacagttgtc aagactgttt
gtcgggtttc caatgctcac 1140acagggtgat aaacaatgct gatacgaaca
catgtgtttc gaaataggct ttgttcttgg 1200tttaatgaag ttacatgtaa
catctactta tattcagatt taaacaatgc ccgcctcaac 1260tgagataacg
gacatccatc aagctcaaaa agaaaacttg tgtttcccat agtcacaaaa
1320gattgcggtt tgtatttcgc atagccaccg tttccgtagt aaaaaacggt
tgcggtttgt 1380atttcgcata gccaccgttt ccgtagtaaa aaacggttgg
gtttgcacat tcaatcttca 1440atacttcatt aaaataccac tagtagaaga
acccgacacg tcttcatgtt acggagaatg 1500ttctacaaat tgaaggaaaa
ggaaaagatt tgttgaaatc gaatttgaca actatgacag 1560cttattaaaa
gtagcaatca cctccaatca tttcctggtc aaatcacaca ccataaagtt
1620taaagttatc gtgtgttgta gtttggaaag atgaccgaac aaagatctga
ataacaaact 1680gtggagaact tgtttgtgtc gggttcttct gctagttact
tgatcaacgg actgatcgtt 1740aacgttactc tacttgtgct ccgatttaag
cggcgtgttc cgatcaacgt tgtttaaagg 1800tgatgaagag tttagaaacc
ctagaatgat ggtgctttta cgggaaaaaa cgtgataatt 1860gaaaaacatc
aatctatacg gcctgtatac aattatacgg cccgtataca tttggtaaac
1920atcaaaaacc tcccaaaata aatccattga gccgtatagt ttttacaaat
cgtatacaca 1980tgtatacggc tcgtataact atatacgggc cgtatatagt
aaaaaaaaca ttctttacgc 2040attgtcctat taaaaaacat tctatacgga
ccgtatattt tgtatacggt ccgtataaac 2100taaagatatg aaaataaaat
atagggggtg tgggaataag aggagcgtta ttaaaataca 2160caaataattt
tcgtcatgac tccaactaca tgttttattg tgttcttgtt tgtcgttatt
2220aatattttct ttgacataga aagtcgaaac gcatggtatc cagctacgta
cacatttatt 2280caacttatat ttattttagt cctcgtttga ctttatgcct
ccactagctt tctccatctg 2340tttatcacgc
tgcaaataag tcaaacaatt ctccaccgtc cgatcaaaac agtatctcat
2400tgactccact ttcccaattg gacctttata acccccttct acccctcata
tctctcatca 2460atctctcttt ctctctctta gggtttcaac ttccaacctt
cttctacaac a 251111817DNAHelianthus annuus 11atggcggttg atttcgtcgg
aattcaatct accgatcatc ttctaaaccg catgttccag 60ttattaagtc acgatttaaa
cgtttcgtca acctacacgc acgcggtttc tgctttcaaa 120cgcaccggtc
acgcacggtt ccgccgtgga ccgtcgtcta ccaccggaga cactaacgga
180ccttcaactt cttcacattc ggaaggtaaa tcacgagatg cgacttcgtt
tgtacaaaac 240gagtgttttt caaacaaacc ggtgacggag ataacgacga
cgacgacgtc aacgagctcg 300tcgtctgtag tatcgtcttc caccggtgga
aacttagacg gaagtgtttc caacggtaaa 360cagttttctt cgttaggtat
agtagctccg gcgccgacgt tctcgtctag aaaaccaccg 420ttaccgtcga
cacaccggaa aaggtgcggc gctgatcgtc ctgttgcttc cgtacacgga
480tccggaagcg gttgccattg ttgttccaag agaaggaaaa ccggatctaa
acgtgaaatt 540agaagagttc cgattaccgg atctaaaatt acaagcatac
ctgctgatga ttactcatgg 600aaaaagtacg gcgagaagaa gatcgacggt
tcactttatc cacgagtata ttacaaatgt 660attaccggaa aaggatgtcc
ggcgaggaag cgcgtggagt taagcgccga cgattcgaag 720atgcttattg
ttacttacga cggagaacac cgtcaccgtg accgtcacgc gccggtacct
780atgagtttga ccggtgtgta tggtgagcca aagtgaa 81712271PRTHelianthus
annuus 12Met Ala Val Asp Phe Val Gly Ile Gln Ser Thr Asp His Leu
Leu Asn1 5 10 15Arg Met Phe Gln Leu Leu Ser His Asp Leu Asn Val Ser
Ser Thr Tyr 20 25 30Thr His Ala Val Ser Ala Phe Lys Arg Thr Gly His
Ala Arg Phe Arg 35 40 45Arg Gly Pro Ser Ser Thr Thr Gly Asp Thr Asn
Gly Pro Ser Thr Ser 50 55 60Ser His Ser Glu Gly Lys Ser Arg Asp Ala
Thr Ser Phe Val Gln Asn65 70 75 80Glu Cys Phe Ser Asn Lys Pro Val
Thr Glu Ile Thr Thr Thr Thr Thr 85 90 95Ser Thr Ser Ser Ser Ser Val
Val Ser Ser Ser Thr Gly Gly Asn Leu 100 105 110Asp Gly Ser Val Ser
Asn Gly Lys Gln Phe Ser Ser Leu Gly Ile Val 115 120 125Ala Pro Ala
Pro Thr Phe Ser Ser Arg Lys Pro Pro Leu Pro Ser Thr 130 135 140His
Arg Lys Arg Cys Gly Ala Asp Arg Pro Val Ala Ser Val His Gly145 150
155 160Ser Gly Ser Gly Cys His Cys Cys Ser Lys Arg Arg Lys Thr Gly
Ser 165 170 175Lys Arg Glu Ile Arg Arg Val Pro Ile Thr Gly Ser Lys
Ile Thr Ser 180 185 190Ile Pro Ala Asp Asp Tyr Ser Trp Lys Lys Tyr
Gly Glu Lys Lys Ile 195 200 205Asp Gly Ser Leu Tyr Pro Arg Val Tyr
Tyr Lys Cys Ile Thr Gly Lys 210 215 220Gly Cys Pro Ala Arg Lys Arg
Val Glu Leu Ser Ala Asp Asp Ser Lys225 230 235 240Met Leu Ile Val
Thr Tyr Asp Gly Glu His Arg His Arg Asp Arg His 245 250 255Ala Pro
Val Pro Met Ser Leu Thr Gly Val Tyr Gly Glu Pro Lys 260 265 270
* * * * *